Microbiological production of 3-hydroxyisobutyric acid

ABSTRACT

The present invention relates to a cell which has been modified in comparison with its wild type in such a way that it is capable of forming more, by comparison with its wild, 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via methylmalonate-semialdehyde or 3-hydroxybutyryl-coenzyme A as precursors. The invention also relates to a method of generating a genetically modified cell, to the genetically modified cell obtainable by these methods, to a method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, to a method of producing methacrylic acid or methacrylic esters, and to a method of producing polymethacrylic acid or polymethacrylic esters. The present invention furthermore relates to an isolated DNA, to a vector, to the use of this vector for transforming a cell, to a transformed cell, and to a polypeptide.

This application is a Divisional of U.S. application Ser. No. 12/303,161, filed Apr. 6, 2009, which is the U.S. National Stage of International Application No. PCT/EP2007/0055394, filed Jun. 1, 2007, which claimed priority to German application no. 102006025821.5, filed Jun. 2, 2006, of which all of the disclosures are incorporated herein by reference in their entireties.

The present invention relates to cells which have been genetically modified in comparison with their wild type, to methods of generating a genetically modified cell, to the genetically modified cells obtainable by these methods, to a process for the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, to a process for the preparation of methacrylic acid or methacrylic esters, and to a process for the preparation of polymethacrylic acid or polymethacrylic esters. The present invention furthermore relates to an isolated DNA, to a vector, to the use of this vector for the transformation of a cell, to a transformed cell, and to a polypeptide.

Methacrylic acid is an important intermediate which is employed for the preparation of polymers, in particular in the form of its alkyl esters. An example of a well-known methacrylic acid derivative is the methyl ester of methacrylic acid. The current global annual production of methyl methacrylate amounts to approximately 1.5 million tonnes. The polymethacrylic esters are raw materials in the plastics sector with a multiplicity of uses.

Methacrylic acid is usually produced commercially by means of the heterogeneous gas-phase oxidation of C₄-carbon compounds such as butylene, isobutylene, butane, isobutane, t-butyl alcohol or methacrolein by two-step catalysis on solid multi-metal oxide compositions as the catalyst. The resulting product gas mixture, which, besides methacrylic acid, also comprises a large number of secondary products, is subsequently either subjected to a total condensation reaction, generating aqueous methacrylic acid solution, or absorbed in a suitable solvent mixture. This is usually followed by further purification of the resulting liquid phases by means of distillation, crystallization, extraction, or a combination of these measures. Besides the catalytic gas-phase oxidation of C₄-carbon compounds, methacrylic acid can also be formed from isobutyric acid by catalytic oxidative dehydrogenation, as is described for example in EP-A-0 356 315. A further possibility for preparing methacrylic acid is what is known as the “ACH process”, in which acetone cyanohydrin and sulfuric acid are reacted with the formation of methacrylamide as intermediate, which then reacts further with water to give methacrylic acid. The resulting methacrylic acid is subsequently purified by distillation. This process is described for example in EP-A-1 359 137.

The disadvantage of these conventional processes for the preparation of methacrylic acid is, inter alia, that during both the preparation of the methacrylic acid itself and during the subsequent steps, which involve purification by distillation, the process steps, which cause thermal stress, result, owing to the pronounced susceptibility of methacrylic acid to polymerization, in the formation of dimers or oligomers; this not only entails additional purification efforts, but also yield losses.

It was an object of the present invention to overcome the disadvantages of the prior art.

In particular, it was an object of the present invention to provide a process for the preparation of methacrylic acid which generates methacrylic acid with a minimum of steps which involve thermal stress.

Furthermore, it is intended that this process makes possible the preparation of methacrylic acid from renewable resources, in particular from carbohydrates and/or glycerol.

BRIEF DESCRIPTIONS OF THE SEVERAL VIEWS OF DRAWINGS

FIG. 1 shows the conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A with catalysis by the enzyme E₁.

FIG. 2 shows the conversion of methylmalonyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₂ to E₄ in accordance with the first alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 3 shows the conversion of (R)-methylmalonyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₄, E₆ and E₇ in accordance with the second alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3 hydroxyisobutyric acid.

FIG. 4 shows the conversion of methylmalonyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₄, E₅ and E₇ in accordance with the third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 5 shows the conversion of 3-hydroxyisobutyric acid into a polyhydroxyalkanoate with catalysis by the enzymes E₈ and E₉.

FIG. 6 shows the conversion of phosphoenolpyruvate or pyruvate into oxalacetate with catalysis by the enzymes E₁₀ or E₁₁ according to a special embodiment of the first, second or third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 7 shows the conversion of oxalacetate into succinyl-coenzyme A with catalysis by the enzymes E₁₂ to E₁₅ according to a first special embodiment of the first, second or third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 8 shows the conversion of oxalacetate into succinyl-coenzyme A with catalysis by the enzymes E₁₃ to E₁₆ and E₂₄ to E₂₆ according to a second special embodiment of the first, second or third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 9 shows the conversion of oxalacetate into succinyl-coenzyme A with catalysis by the enzymes E₁₆, E₂₄, E₂₇ and E₂₈ according to a third special embodiment of the first, second or third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 10 shows the conversion of L-glutamate into succinyl-coenzyme A with catalysis by the enzymes E₄₆ and E₂₈ in accordance with a further special embodiment of the first, second or third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 11 shows the conversion of acetyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₄, E₅ and E₄₇ to E₅₂ in accordance with a first alternative of the second special embodiment of the cell according to the invention, where propionyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 12 shows the conversion of propionyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₂ to E₄, E₆, E₇ and E₄₇ to E₅₂ in accordance with a second alternative of the second special embodiment of the cell according to the invention, where propionyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 13 shows the conversion of propionyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₂ to E₄, E₇ and E₄₇ to E₅₂ in accordance with a third alternative of the second special embodiment of the cell according to the invention, where propionyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 14 shows the conversion of propionyl-coenzyme A into 3-hydroxyisobutyric acid with catalysis by the enzymes E₂ to E₄, E₇ and E₄₇ to E₅₂ in accordance with a fourth, fifth alternative of the second special embodiment of the cell according to the invention, where propionyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 15 shows the conversion of β-alanine into 3-hydroxyisobutyric acid with catalysis by the enzymes E₁₀ to E₁₂, E₅₆, E₇₂ and E₇₃ according to a third special embodiment of the cell according to the invention, where acrylyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 16 shows the conversion of pyruvate into 3-hydroxyisobuytric acid with catalysis by the enzymes E₇₅ to E₇₉, E₆₀, E₆₁ and E₈ according to a first alternative of the first special embodiment of the second variant of the cell according to the invention, where isobutyryl-coenzyme A is formed as intermediate and 3-hydroxyisobutyryl-coenzyme A as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 17 shows the conversion of L-valine into 3-hydroxyisobutyric acid with catalysis by the enzymes E₈, E₆₀, E₆₁, E₇₉ and E₈₀ according to a second alternative of the first special embodiment of the second variant of the cell according to the invention, where isobutyryl-coenzyme A is formed as intermediate and 3-hydroxyisobutyryl-coenzyme A as precursor in the production of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid.

FIG. 18 shows the conversion of beta-Alanine to 3 hydroxyisobutyric acid.

FIG. 19 shows the conversion of (S)-2-Acetolactate to methylacrylyl-coenzyme A.

FIG. 20 shows the conversion of L-valine to 3 hydroxyisobutyric acid.

A contribution to achieving the abovementioned aims is provided by a cell which has been genetically modified in comparison with its wild type in such a way that it is capable of forming more 3-hydroxyisobutyric acid, or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, but preferably more 3-hydroxyisobutyric acid, in comparison with its wild type, this formation preferably taking place via methylmalonate semialdehyde or via 3-hydroxyisobutyryl-coenzyme A as precursor.

In the event that the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor, it is furthermore preferred that the formation takes place via succinyl-coenzyme A, propionyl-coenzyme A or acryloyl-coenzyme A, especially preferably via succinyl-coenzyme A, as further intermediate. In the event that the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor, it is furthermore preferred that the formation takes place via isobutyryl-coenzyme A or via 3-hydroxybutyryl-coenzyme A, preferably via 3-hydroxybutyryl-coenzyme A, as further intermediate.

The term “precursor” as used in the present context defines a chemical compound which can be converted enzymatically into 3-hydroxyisobutyric acid in just one reaction step, while the term “intermediate” defines a chemical compound which cannot be converted enzymatically into 3-hydroxyisobutyric acid in just one reaction step.

The term “3-hydroxyisobutyric acid” as used in the present context always describes the corresponding C₄-carboxylic acid in the form in which it is present as a function of the pH, after having been formed by the microorganisms in question. As a consequence, the term always comprises the pure acid form (3-hydroxyisobutyric acid), the pure base form (3-hydroxyisobutyrate) and mixtures of protonated and deprotonated forms of the acid. Furthermore, the term “3-hydroxyisobutyric acid” comprises, in principle, both the (R) and the (S) stereoisomer, the (S) stereoisomer being especially preferred.

The wording “that it is capable of forming more 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid in comparison with its wild type” also applies in the event that the wild type of the genetically modified cell is not capable of forming any 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, but at least no detectable amounts of these compounds, and that detectable amounts of these components are only capable of being formed after the genetic modification.

A “wild type” of a cell preferably refers to a cell whose genome is present in a state as generated naturally as the result of evolution. The term is used both for the entire cell and for individual genes. As a consequence, the term “wild type” does not cover in particular those cells, or those genes, whose gene sequences have at least in part been modified by man by means of recombinant methods.

The 3-hydroxyisobutyric acid can subsequently give rise to methacrylic acid by subjecting it to a dehydration reaction under mild conditions. In the case of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, the vesicles present in the cells, which are filled with these polyhydroxyalkanoates, can be isolated and the polymers can subsequently be cleaved to give 3-hydroxyisobutyric acid, which can then be dehydrated to give methacrylic acid.

In this context, it is preferred according to the invention that the genetically modified cell has been genetically modified in such a way that it forms at least twice, especially preferably at least 10 times, more preferably at least 100 times, even more preferably at least 1000 times and most preferably at least 10 000 times more 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid than the wild type of the cell within a defined time interval, preferably within 2 hours, even more preferably within 8 hours and most preferably within 24 hours. The increase in the formation of product can be determined in this context for example by growing the cell according to the invention and the wild-type cell in each case separately, but under identical conditions (identical cell density, identical nutrient medium, identical culture conditions) for a particular time interval in a suitable nutrient medium and subsequently determining the amount of target product (3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid) in the nutrient medium.

The cells according to the invention may be prokaryotic or eukaryotic cells. They may take the form of mammalian cells (such as, for example, human cells), of plant cells or of microorganisms such as yeasts, fungi or bacteria, with microorganisms being especially preferred and bacteria and yeasts being most preferred.

Suitable bacteria, yeasts or fungi are in particular those bacteria, yeasts or fungi which have been deposited at the Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSMZ), Brunswick, Germany, as bacterial, yeast or fungal strains. Bacteria which are suitable according to the invention belong to the genera detailed under

http://www.dsmz.de/species/bacteria.htm, yeasts which are suitable according to the invention belong to those genera which are detailed under http://www.dsmz.de/species/yeasts.htm, and fungi which are suitable according to the invention are those which are detailed under http://www.dsmz.de/species/fungi.htm.

Cells which are especially preferred according to the invention are those of the genera Corynebacterium, Brevibacterium, Bacillus, Acinetobacter, Lactobacillus, Lactococcus, Candida, Pichia, Kluveromyces, Saccharomyces, Escherichia, Zymomonas, Yarrowia, Methylobacterium, Ralstonia, Pseudomonas, Burkholderia and Clostridium, with Brevibacterium flavum, Brevibacterium lactofermentum, Escherichia coli, Saccharomyces cerevisiae, Kluveromyces lactis, Candida blankii, Candida rugosa, Corynebacterium glutamicum, Corynebacterium efficiens, Zymonomas mobilis, Yarrowia lipolytica, Methylobacterium extroquens, Ralstonia eutropha, especially Ralstonia eutropha H16, Rhodospirillum rubrum, Rhodobacter sphaeroides, Para coccus versutus, Pseudomonas aeroginosa, Acinetobacter calcoaceticus and Pichia pastoris being especially preferred.

In accordance with a first variant of the cell according to the invention, the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor.

In accordance with a first special embodiment of this first variant of the cell according to the invention, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid preferentially takes place via succinyl-coenzyme A as intermediate, where the cell preferentially is capable of utilizing carbohydrates, glycerol or glutamate as the carbon source.

Here, it may be advantageous in the context of the first special embodiment of the first variant of the cell according to the invention that the cell according to the invention features an increased activity of an enzyme E₁, which catalyzes the conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A, in comparison with its wild type (see FIG. 1).

The term “increased activity of an enzyme” as used above in connection with the enzyme E₁ and in what follows in the context of the enzymes E₂ etc. is preferably to be understood as increased intracellular activity.

What now follows on increasing the enzymatic activity in cells applies both to increasing the activity of the enzyme E₁ and to all enzymes mentioned thereafter, whose activity can, if appropriate, be increased.

In principle, an increase in the enzymatic activity can be achieved by increasing the copy number of the gene sequence(s) which code for the enzyme, by using a strong promoter or by using a gene or allele which codes for a corresponding enzyme with an increased activity, and, if appropriate, combining these measures. Cells which have been genetically modified in accordance with the invention are generated for example by transformation, transduction, conjugation or a combination of these methods with a vector which comprises the desired gene, an allel of this gene or parts thereof, and a vector which makes possible the expression of the gene. The heterologous expression is achieved in particular by integration of the gene, or of the alleles, into the chromosome of the cell or into an extrachromosomally replicating vector.

An overview over the possibilities for increasing the enzymatic activity in cells with pyruvate carboxylase by way of example is found in DE-A-100 31 999, which is hereby incorporated by reference and whose disclosure content regarding the possibilities for increasing the enzymatic activity in cells forms part of the disclosure of the present invention.

The expression of the enzymes or genes mentioned hereinabove and in each case hereinbelow can be detected in the gel with the aid of 1- and 2-dimensional protein gel separation and subsequent visual identification of the protein concentration using suitable evaluation software. When the increase in an enzymatic activity is based exclusively on an increase in the expression of the gene in question, the quantification of the increase in the enzymatic activity can be determined in a simple manner by comparing the 1- or 2-dimensional protein separations between the wild type and the genetically modified cell. A conventional method of preparing the protein gels in coryneform bacteria, and of identifying the proteins, is the procedure described by Hermann et al. (Electrophoresis, 22: 1712.23 (2001)). The protein concentration can also be analyzed by Western blot hybridization using an antibody which is specific for the protein to be detected (Sambrook et al., Molecular Cloning: a laboratory manual, 2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989) followed by visual evaluation with suitable software for determining the concentration (Lohaus and Meyer (1989) Biospektrum, 5: 32-39; Lottspeich (1999), Angewandte Chemie 111: 2630-2647). The activity of DNA-binding proteins can be measured by means of DNA band shift assays (also referred to as gel retardation) (Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155). The effect of DNA-binding proteins on the expression of other genes can be detected by various, extensively described methods of the reporter gene assay (Sambrook et al., Molecular Cloning: a laboratory manual, 2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. USA, 1989). The intracellular enzymatic activities can be detected by various methods which have been described (Donahue et al. (2000) Journal of Bacteriology 182 (19): 5624-5627; Ray et al. (2000) Journal of Bacteriology 182 (8): 2277-2284; Freedberg et al. (1973) Journal of Bacteriology 115 (3): 816-823). In the event that no specific methods for determining the activity of a particular enzyme are detailed in what follows, the determination of the increase in the enzymatic activity, and also the determination of the reduction in an enzymatic activity, is preferably carried out by means of the methods described in Hermann et al., Electrophoresis, 22: 1712-23 (2001), Lohaus et al., Biospektrum 5 32-39 (1998), Lottspeich, Angewandte Chemie 111: 2630-2647 (1999) and Wilson et al. (2001) Journal of Bacteriology, 183: 2151-2155 (2001).

If increasing the enzymatic activity is brought about by mutating the endogenous gene, such mutations can be generated either undirected, using traditional methods such as for example by UV irradiation or by mutagenic chemicals, or directed by means of recombinant methods such as deletion(s), insertion(s) and/or nucleotide substitution(s). These mutations give rise to genetically modified cells. Especially preferred mutants of enzymes are in particular also those enzymes which are no longer capable of being feedback-inhibited, or which are at least less capable of being feedback-inhibited, in comparison with the wild-type enzyme.

If increasing the enzymatic activity is brought about by increasing the expression of an enzyme, then, for example, the copy number of the respective genes are increased, or the promoter and regulatory regions or the ribosomal binding site, which is located upstream of the structural gene, are mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same manner. By means of inducible promoters it is additionally possible to increase the expression at any desired point in time. Furthermore, the enzyme gene may also have assigned to it what are known as enhancer sequences as regulatory sequences; these also bring about an increased gene expression via an improved interaction between RNA polymerase and DNA. Measures for extending the life of the mRNA also improves expression. Furthermore, preventing the degradation of the enzyme protein also enhances the enzymatic activity. Here, the genes or gene constructs are either present in plasmids in different copy numbers, or else they are integrated and amplified in the chromosome. As an alternative, overexpression of the genes in question may also be achieved by modifying the media composition and the control of the culture.

Instructions for doing so can be found by the skilled worker in Martin et al. (Bio/Technology 5, 137-146 (1987)) in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in EP-A-0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pühler ((Bio/Technology 9, 84-87 (1991), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in WO-A-96/15246, in Malumbres et al. (Gene 134, 15-24 (1993), in JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), inter alia, and in known textbooks of genetics and molecular biology. The above-described measures give rise to genetically modified cells, as do the mutations.

Plasmids, for example episomal plasmids, are employed for increasing the expression of the genes in question. Suitable plasmids are in particular those which are replicated in coryneform bacteria. A large number of known plasmid vectors such as, for example, pZ1 (Menkel et al., Applied and Environmental Microbiology 64: 549-554 (1989)), pEKEx1 (Eikmanns et al., Gene 107: 69-74 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors such as, for example, those based on pCG4 (U.S. Pat. No. 4,489,160) or pNG2 {Serwold-Davis et al., FEMS Microbiology Letters 66: 119-124 (1990)} or pAG1 (U.S. Pat. No. 5,158,891), may be employed in the same manner.

Others which are suitable are those plasmid vectors with the aid of which the method of amplifying genes by integration into the chromosome can be applied, as has been described for example by Reinscheid et al. (Applied and Environmental Microbiology 60: 126-132 (1994)) for duplicating or amplifying the hom-thrB operon. In this method, the entire gene is cloned into a plasmid vector which is capable of replication in a host (typically Escherichia coli), but not in Corynebacterium glutamicum. Suitable vectors are, for example, pSUP301 (Simon et al, Bio/Technology 1: 784-791 (1983)), pK18mob or pK19mob (Schäfer et al., Gene 145: 69-73 (1994)), pGEM-T (Promega Corporation, Madison, Wis., USA), pCR2.1-TOPO (Shuman, Journal of Biological Chemistry 269: 32678-84 (1994)), pCR®Blunt (Invitrogen, Groningen, the Netherlands), pEM1 (Schrumpf et al., Journal of Bacteriology 173: 4510-4516)) or pBGS8 (Spratt et al., Gene 41: 337-342 (1986)). The plasmid vector, which contains the gene to be amplified, is subsequently transferred into the desired Corynebacterium glutamicum strain by means of conjugation or transformation. The conjugation method is described for example in Schäfer et al., Applied and Environmental Microbiology 60: 756-759 (1994). Transformation methods are described for example in Thierbach et al., Applied Microbiology and Biotechnology 29: 356-362 (1988), Dunican and Shivnan, Bio/Technology 7: 1067-1070 (1989) and Tauch et al., FEMS Microbiology Letters 123: 343-347 (1994). Following homologous recombination by means of a cross-over event, the resulting strain comprises at least two copies of the gene in question.

The wording “an activity of an enzyme E_(x) which is increased in comparison with its wild type” used hereinabove and in what follows is preferably always understood as meaning an activity of the respective enzyme E_(x) which is increased by a factor of at least 2, especially preferably of at least 10, more preferably of at least 100, even more preferably of at least 1000 and most preferably of at least 10 000. Furthermore, the cell according to the invention which features “an activity of an enzyme E_(x) which is increased in comparison with its wild type”, in particular also a cell whose wild type features no, or at least no detectable, activity of this enzyme E_(x) and which only shows a detectable activity of this enzyme E_(x) after increasing the enzymatic activity, for example by means of overexpression. In this context, the term “overexpression”, or the wording “increase in the expression” used in what follows also comprises the case that a starting cell, for example a wild-type cell, features no, or at least no detectable, expression and detectable expression of the enzyme E_(x) is only induced by recombinant methods.

Accordingly, the wording “reduced activity of an enzyme E_(x)” used hereinbelow is understood as meaning an activity which is preferably reduced by a factor of at least 0.5, especially preferably of at least 0.1, more preferably of at least 0.01, even more preferably of at least 0.001 and most preferably of at least 0.0001. The reduction in the activity of a specific enzyme can be obtained for example by directed mutation, by the addition of competitive or non-competitive inhibitors or by other measures for reducing the expression of a specific enzyme which are known to the skilled worker.

In the case of the enzyme E₁, which catalyzes the conversion of succinyl-coenzyme A into methylmalonyl-coenzyme A, this preferably takes the form of a methylmalonyl-coenzyme A mutase (EC 5.4.99.2). This enzyme is preferably encoded by the gene selected from the group consisting of mut, mutA, mutB, sbm, sbmA, sbmB, sbm5, bhbA, mcmA, mcmA1, mcmA2, mcmB, mcm1, mcm2, mcm3, icmA, meaA1 and meaA2. The nucleotide sequence of these genes can be found for example in the “Kyoto Encyclopedia of Genes and Genomes” (KEGG database), the databases of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA) or from the nucleotide sequence database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany and Cambridge, UK).

In accordance with an especially preferred embodiment of the first variant of the cell according to the invention, the enzyme E₁ takes the form of the methylmalonyl-coenzyme A mutase from Corynebacterium glutamicum ATCC 13032, which is encoded by a gene with the DNA sequence as shown in SEQ ID No 01 and which has the amino acid as shown in SEQ ID No 02.

Furthermore, it is preferred in accordance with a first alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, that the cell, if appropriate in addition to the increased activity of the enzyme E₁, features an activity of at least one of the following enzymes E₂ to E₄ which is increased in comparison with its wild type (see FIG. 2):

-   -   of an enzyme E₂, which catalyzes the conversion of         methylmalonyl-coenzyme A into methyl malonate;     -   of an enzyme E₃, which catalyzes the conversion of methyl         malonate into methylmalonate semialdehyde;     -   of an enzyme E₄ which catalyzes the conversion of methylmalonate         semialdehyde into 3-hydroxyisobutyric acid.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₂, E₃, E₄, E₂E₃, E₂E₄, E₃E₄, E₂E₃E₄, where E₂E₃E₄ is most preferred. Furthermore, it is possible that an enzyme is also capable of catalyzing at least two of the above-described reaction steps. Thus, for example, it is possible to employ an enzyme which features both the activity of enzyme E₂ and that of enzyme E₃ (and which therefore catalyzes the conversion of methylmalonyl-coenzyme A directly into methylmalonate semialdehyde) such as, for example, the malonyl coenzyme A reductase from Sulfolobus tokodaii, which is encoded by the DNA sequence with the SEQ ID No 03 and which has the amino acid sequence as shown in SEQ ID No 04, or else an enzyme which features all three enzymatic activities E₂, E₃ and E₄, such as the malonyl coenzyme A reductase from Chloroflexus aurantiacus (Hügler et al., Journal of Bacteriology 184, pages 2404-2410, 2002).

In this context, it is especially preferred that the enzyme

-   E₂ is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17), -   E₃ is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase     (EC 1.2.3.1) and -   E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a     3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

The enzyme E₂ is preferably encoded by the aox1 gene. The methylmalonyl-coenzyme A hydrolase from rat liver is described for example in Kovachy et al., “Recognition, isolation, and characterization of rat liver D-methylmalonyl coenzyme A hydrolase”, J. Biol. Chem. 258 (1983), pages 11415-11421.

The enzyme E₃ is preferably encoded by genes selected from the group consisting of aldh2, aldh3a1, aldh3a2, aldh1b1, aldh9a1, aldh1a1, aldh1a4, aldh1a1, aldh1a2, mgc80785, mgc83352, mgc89020, dmel-CG31075, cg3752, cg9629, alh-9, alh-1, alh-2, f5O8.35, t7O23.15, f15I1.19, tT17F15.130, ald1, ald2, ald4, ald5, ald6, ac1044Wp, adr417wp, msc7, tb06.5F5.780, aldH, puuC, putA, aldA, badH, alkH, pcD, rsp1591, rs01031, exaC, acoD, dhaL, pchA, aldB, dhaS, betB, ywdH, ycbD, aldX, aldY, aldA1, aldA2, aldC, pcd, cg10546, cg12668, cg12796, scg11A.05, sci30A.27c, sce9.27c, sck13.05c, sc5H4.03, thcA, gabD2, alkH, aldH, aldH1, aldA1, aldY2, aldY3, aldY4, aldY5, aldY6, aldY7 and aldhT.

Suitable genes for the enzyme E₄ are selected from the group consisting of hibadh, cg15093, cg15093, cg4747, mwL2.23, t13k14.90, f19b15.150, hibA, ygbJ, mmsB, mmsB, garR, tsar, mmsB-1, mmsB-2, yfjR, ykwC, ywjF, hibD, glxR, SCM1.40c, hibD, ehhand, hadh2, hadhsc, hsd17B4, loc488110, had, mgC81885, hadh2-prov, cg3415, cg7113, ech-1, ech-8, ech-9, ard-1, yfcX, fadB, faoA, fadB2x, hbd-1, hbd-2, hbd-3, hbd-4, hbd-5, hbd-6, hbd-7, hbd-8, hbd-9, hbd-10, fadJ, rs04421, rs02946, rs05766, bbsD, bbsC, fadB1, fadB2, fadB5, hbdA, pimF, fabJ-1, fabJ, scbac19f3.11, sci35.13, scbac8d1.10c, sc5f2a.15, sc6a5.38, fadC2, fadC4, fadC5, fadC6, had and paaH. Further suitable 3-hydroxyisobutyrate dehydrogenases are described for example in Bannerjee et al. (1970), J. Biol. Chem, 245, pages 1828 to 1835, Steele et al. (1992), J. Biol. Chem., 267, pages 13585 to 13592, Harris et al. (1988), J. Biol. Chem., 263, pages 327 to 331, Harris et al., Biochim. Biophys. Acta, 1645 (1), pages 89 to 95, Hawes et al. (2000), Methods Enzymol., 324, pages 218 to 228, Harris et al., J. Biol. Chem., 275 (49), pages 38780 to 38786, Rougraff et al. (1988), J. Biol. Chem., 263(1), pages 327 to 331, Robinson et al., J. Biol. Chem., 225, pages 511 to 521, Hawes et al. (1995), Biochemistry, 34, pages 4231 to 4237, Hasegawa J. (1981), Agric. Biol. Chem., 45, pages 2805 to 2814, Hawes et al. (1996), FEBS Lett., 389, pages 263 to 267, Hawes et al. (1996), Enzymology and Molecular Biology of Carbonyl Metabolism, Plenum Press, New York, pages 395 to 402, Adams et al. (1994), Structure, 2, pages 651 to 668, Zhang et et. (1999), Biochemistry, 38, pages 11231 to 11238, Mirny et al., (1999), J. Mol. Biol., 291, pages 177 to 196 and Lokanath et al. (2005), J Mol Biol. The disclosure of these publications is hereby incorporated by reference and forms part of the disclosure of the present invention.

The nucleotide sequences of the abovementioned genes and of further genes for the enzymes E₂ to E₄ can also be found in the KEGG database, the NCBI database or the EMBL database, inter alia.

In accordance with an especially preferred embodiment of this alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, it is preferred that the malonyl coenzyme A reductase from Sulfolobus tokodaii, which is encoded by the DNA sequence with the SEQ ID No and which has the amino acid sequence as shown in SEQ ID No 04, is employed for the conversion of methylmalonyl-coenzyme A into methylmalonate semialdehyde. In accordance with another especially preferred embodiment of this variant, the malonyl coenzyme A reductase from Chloroflexus aurantiacus (Hügler et al., Journal of Bacteriology 184, pages 2404-2410, 2002) is employed for the conversion of methylmalonyl-coenzyme A into 3-hydroxyisobutyric acid.

Furthermore, it is preferred in the context of this first alternative of the first special embodiment of the cell according to the invention that the cell features an activity of an enzyme E₅, which features the conversion of methylmalonate semialdehyde into propionyl-coenzyme A, which is reduced in comparison with its wild type, this enzyme preferably taking the form of a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27).

In accordance with a second alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, it is preferred that the cell, if appropriate in addition to the increased activity of the enzyme E₁, features an activity of at least one of the following enzymes E₄ to E₇ which is increased in comparison with its wild type (see FIG. 3):

-   -   of an enzyme E₆, which catalyzes the conversion of (R)         methylmalonyl-coenzyme A into (S) methyl-malonyl-coenzyme A;     -   of an enzyme E₇, which catalyzes the conversion of (S)         methylmalonyl-coenzyme A into propionyl-coenzyme A;     -   of an enzyme E₅, which catalyzes the conversion of         propionyl-coenzyme A into methylmalonate semialdehyde;     -   of an enzyme E₄, which catalyzes the conversion of         methylmalonate semialdehyde into 3-hydroxyisobutyric acid.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₄, E₅, E₆, E₇, E₇, E₄E₅, E₄E₆, E₄E₇, E₅E₆, E₅E₇, E₆E₇, E₄E₅E₆, E₄E₅E₇, E₄E₆E₇, E₅E₆E₇ and E₄E₅E₆E₇, with E₄E₅E₆E₇ being most preferred.

In this context, it is especially preferred that the enzyme

-   E₆ is a methylmalonyl-coenzyme A epimerase (EC 5.1.99.1) -   E₇ is a methylmalonyl-coenzyme A decarboxylase (EC 4.1.1.41), -   E₅ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), and -   E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a     3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

In this context, preferred enzymes E₄ are those which have already been mentioned above in the context of the first variant of the first preferred embodiment of the cell according to the invention.

The enzyme E₆ is preferably encoded by the mcee gene. A suitable methylmalonyl-coenzyme A decarboxylase (enzyme E₇) is described, for example, by Benning et al. in Biochemistry, Vol. 39 (2000), pages 4630-4639.

Suitable genes for the enzyme E₅ are preferably selected from the group consisting of aldh6a1, cg17896, t22c12.10, ald6, putA1, mmsA, mmsA-1, mmsA-2, mmsA-3, mmsA-4, msdA, iolA and iolAB.

Suitable genes for the enzyme E₇ are preferably selected from the group consisting of mmdA, bcc, oadB, oadB2, oadB3, SC1C2.16, SC1G7.10, pccB1, accA2, mmdB, mmdC and ppcB.

The nucleotide sequences of the abovementioned genes for the enzymes E₅, E₆ and E₇ may, inter alia, also be found in the KEGG database.

In accordance with a third alternative of the cell according to the invention, where succinyl-coenzyme A is formed as intermediate and methylmalonate semialdehyde as precursor in the preparation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, it is preferred that the cell, if appropriate in addition to the increased activity of the enzyme E₁, features an activity of at least one of the following enzymes E₄, E₅ and E₇ which is increased in comparison with its wild type (see FIG. 4):

-   -   of an enzyme E₇, which catalyzes the conversion of         methylmalonyl-coenzyme A into propionyl-coenzyme A;     -   of an enzyme E₅, which catalyzes the conversion of         propionyl-coenzyme A into methylmalonate-semialdehyde;     -   of an enzyme E₄, which catalyzes the conversion of         methylmalonate-semialdehyde into 3-hydroxyisobutyric acid.

This pathway corresponds essentially to the second variant of the first preferred embodiment of the cell according to the invention, but, as opposed to the second variant, propionyl-CoA is prepared directly from methylmalonyl-coenzyme A. Preferred enzymes and genes for the enzymes E₄, E₅ and E₇ are those genes or enzymes which have already been mentioned above in connection with the second variant.

Furthermore, it may in accordance with the first special embodiment of the cell according to the invention (and also in accordance with all embodiments which are still to be described hereinbelow) also be preferred that the cell is capable of converting the formed 3-hydroxyisobutyric acid into a polyhydroxyalkanoate. Such polyhydroxydalkanoates are deposited intracellularly by many microorganisms in the form of highly refractive granula. In this context, it is especially preferred that the cell according to the invention features an activity of at least one of, preferably of the two, the following enzymes E₉ and E₁₀ which is increased in comparison with its wild type (see FIG. 5):

-   -   of an enzyme E₈, which catalyzes the conversion of         3-hydroxyisobutyric acid into 3-hydroxyisobutyryl-coenzyme A;     -   of an enzyme E₉, which catalyzes the conversion of         3-hydroxyisobutyryl-coenzyme A to a polyhydroxyalkanoate based         on 3-hydroxyisobutyric acid.

In this context, it is especially preferred that the enzyme

-   E₈ is a 3-hydroxyisobutyryl CoA hydrolase (EC 3.1.2.4) and -   E₉ is a polyhydroxyalkanoate synthase.

As has already been explained above, the first preferred embodiment of the cell according to the invention generates 3-hydroxyisobutyric acid or the polyhydroxyalkanoates based on 3-hydroxyisobutyric acid from succinyl coenzyme A as intermediate and from methylmalonate semialdehyde as precursor. Here, it may make sense in principle to influence not only one or more of the abovementioned enzyme activities E₁ to E₉, but also those enzyme activities which lead to an increased formation of succinyl-coenzyme A in the cell.

In the event that, according to the first special embodiment of the first variant of the cell according to the invention, the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place from carbohydrates or glycerol via succinyl-coenzyme A as intermediate and methylmalonate semialdehyde as precursor, it is, according to a special embodiment of the above-described first, second or third alternative of the cell according to the invention, preferred that the cell features an activity of at least one of the, preferably of the two, following enzymes E₁₀ and E₁₁ which is increased in comparison with its wild type (see FIG. 6):

-   -   of an enzyme E₁₀, which catalyzes the conversion of         phosphoenolpyruvate into oxaloacetate;     -   of an enzyme E₁₁, which catalyzes the conversion of pyruvate         into oxaloacetate.

In this context, it is especially preferred that the enzyme

-   E₁₀ is a phosphoenolpyruvate carboxylase (EC 4.1.1.31) and -   E₁₁ is a pyruvate carboxylase (EC 6.4.1.1).

The enzyme E₁₀ is preferably encoded by the genes selected from the group consisting of f12m16.21, f14n22.13, k15m2.8, ppc, clpA, pepC, capP, cgl1585, pepC, pck ppc and pccA, where the ppc gene is especially preferred. Phosphoenolpyruvate carboxylases which are preferred according to the invention are also described in particular in U.S. Pat. No. 4,757,009, U.S. Pat. No. 4,980,285, U.S. Pat. No. 5,573,945, U.S. Pat. No. 6,872,553 and U.S. Pat. No. 6,599,732. As regards phosphoenolpyruvate carboxylases, the disclosure content of these publications is hereby incorporated by reference and forms part of the disclosure of the present invention.

The enzyme E₁₁ is preferably encoded by the genes selected from the group consisting of pc, pcx, cg1516, cg1516, pyc-1, pyc-2, aar162Cp, pyr1, accC-2, pycA, pycA2, pca, cgl0689, pyc, pycB, accC, oadA, acc and accC1, where the pyc gene is especially preferred. Pyruvate carboxylases which are preferred according to the invention are also described in particular in U.S. Pat. No. 6,455,284, U.S. Pat. No. 6,171,833, U.S. Pat. No. 6,884,606, U.S. Pat. No. 6,403,351, U.S. Pat. No. 6,852,516 and U.S. Pat. No. 6,861,246. A further pyruvate carboxylase which is especially preferred according to the invention is that mutant which is described in “A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant”, Ohnishi J et al., Applied Microbiology and Biotechnology, vol. 58 (2), pages 217-223 (2002).

The nucleotide sequences of suitable genes of the enzymes E₁₁ and E₁₂ can be found in the KEGG database, the NCBI database or the EMBL database.

Starting from the oxaloacetate intermediate stage, there are several possibilities for arriving at succinyl-coenzyme A, which can then be converted into 3-hydroxyisobutyric acid via methylmalonyl-coenzyme A by means of the three variants mentioned at the outset.

A first pathway leads via fumarate as intermediate. In this case it is preferred in accordance with a first special embodiment of the above-described first, second or third alternative of the cell according to the invention, where methylmalonate-semialdehyde is formed as precursor and succinyl-coenzyme A as intermediate, that the cell, if appropriate additionally to an increased activity of the enzyme E₁₀ or E₁₁, features an activity of at least one of the following enzymes E₁₂ to E₁₅ which is increased in comparison with its wild type (see FIG. 7):

-   -   of an enzyme E₁₂, which catalyzes the conversion of oxaloacetate         into malate;     -   of an enzyme E₁₃, which catalyzes the conversion of malate into         fumarate;     -   of an enzyme E₁₄, which catalyzes the conversion of fumarate         into succinate;     -   of an enzyme E₁₅, which catalyzes the conversion of succinate         into succinyl-coenzyme A.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₁₂, E₁₃, E₁₄, E₁₅, E₁₂E₁₃, E₁₂E₁₄, E₁₂E₁₅, E₁₃E₁₄, E₁₃E₁₅, E₁₄E₁₅, E₁₂E₁₃E₁₄, E₁₂E₁₃E₁₅, E₁₂E₁₄E₁₅, E₁₃E₁₄E₁₅, E₁₂E₁₃E₁₄E₁₅, with E₁₂E₁₃E₁₄E₁₅ being most preferred.

In this context, it is especially preferred that the enzyme

-   E₁₂ is a malate dehydrogenase (EC 1.1.1.37) or a malate quinone     oxidoreductase (1.1.99.16), -   E₁₃ is a fumarate hydratase (EC 4.2.1.2), -   E₁₄ is a succinate dehydrogenase (EC 1.3.99.1 or EC 1.3.5.1) or a     succinate quinone oxidoreductase (1.3.5.1), and -   E₁₅ is a succinate coenzyme A ligase (EC 6.2.1.4 or EC 6.2.1.5).

The enzyme E₁₂ is preferably encoded by genes selected from the group consisting of mdh1, mdh2, mor1, cg10748, cg10749, cg5362, mdh-1, f46e10.10, f19p19.13, f12m16.14, t30120.4, k15m2.16, f1p2.70, f17i14.150, mn112.18, mik19.17, mdh3, ad1164cp, adr152cp, adr252wp, mdhA, mdhC, mdhB, ybiC, mdh, yiaK, ybiC, allD, citH, yjmC, citH, cgl2380, ldh, sqdB, mqo, yojH, mqoA, mqoB, mqo1, mqo2, mqo3, mqo4 and cgl2001, where the mqo gene and the mdh gene are especially preferred.

The enzyme E₁₃ is preferably encoded by genes selected from the group consisting of fh, fh1, sc4094, sc4095, t30b22.19, k3k7.11, acr013/cp, fum1, fum2, fum3, fum4, fumH, fumA, fumB, fumC, fumC1, fumC2, fum, ttdA, ttdB, fumB-alpha, fumB-beta, citG, citB, fumX, fum-1 and fum-2, where the fum gene is especially preferred.

The enzyme E₁₄ is preferably encoded by genes selected from the group consisting of sdh1, sdh2, sdh3, sdh4, sdh5, sdh6, osm1, osm2, sdhA, sdhB, sdhC, sdhD, frdA, frdB, frdC, frdD, ifcA-1, ifcA-2, sdhB-1, sdhB-2, frdC2, cgl0370, cgl0371, cgl0372, scm10.10c, scm10.11c, scm10.12c, sc5g8.25c, sc5g8.26c, scbac-31e11.02c, scbac31e11.02c, sc4b10.10c, sdhA2, sdhB2, sdhA1, sdhB1, qcrB2, sdhA3, sdhB3, frdB1 and frdB2, where the genes sdhA, sdhB and sdhC are especially preferred.

The enzyme E₁₅ is preferably encoded by genes selected from the group consisting of suclg1, suclg2, loc434885, cg10622, dmel-CG6255, flla3.3, f8115.30, mkd15.11, lsc1, lsc2, ae1211wp, afrl34cp, scsA, scsB, sucC and sucD.

Again, the nucleotide sequences of suitable genes of the enzymes E₁₂ to E₁₅, can also be found in the KEGG database, the NCBI database or the EMBL database.

In the event that the activity of one or more of the enzymes E₁₂ to E₁₅ is increased, it may also prove advantageous that the cell features an activity of one of the following enzymes E₁₆ to E₂₃ which is reduced in comparison with its wild type:

-   -   of an enzyme E₁₆, which catalyzes the conversion of oxaloacetate         into citrate;     -   of an enzyme E₁₇, which catalyzes the conversion of malate into         oxaloacetate;     -   of an enzyme E₁₈, which catalyzes the conversion of         succinyl-coenzyme A into succinate,     -   of an enzyme E₁₉, which catalyzes the conversion of oxaloacetate         into phosphoenolpyruvate, of an enzyme E₂₀, which catalyzes the         conversion of oxaloacetate into pyruvate,     -   of an enzyme E₂₁, which catalyzes the conversion of oxaloacetate         into aspartate,     -   of an enzyme E₂₂, which catalyzes the conversion of malate into         pyruvate,     -   of an enzyme E₂₃, which catalyzes the conversion of pyruvate         into acetate.

Cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is reduced: E₁₆, E₁₇, E₁₈, E₁₉, E₂₀, E₂₁, and E₁₆E₁₇E₁₈E₁₉E₂₀E₂₁E₂₂E₂₃.

In this context, it is especially preferred that the enzyme

-   E₁₆ is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8), -   E₁₇, is a malate oxidase (EC 1.1.3.3), -   E₁₈ is a succinyl CoA hydrolase (EC 3.1.2.3), -   E₁₉ is a phosphoenolpyruvate carboxykinase (EC 4.1.1.49 or     4.1.1.32), -   E₂₀ is an oxaloacetate decarboxylase (EC 4.1.1.3), -   E₂₁ is an aspartate transaminase (EC 2.6.1.1), -   E₂₂ is a malate dehydrogenase (EC 1.1.1.38, EC 1.1.1.39 or EC     1.1.1.40), -   E₂₃ is a pyruvate dehydrogenase (EC 1.2.1.51).

The enzyme E₁₆ is preferably encoded by genes selected from the group consisting of glt, cs, csl, cg3861, cts-1, f7f19.21, f4i1.16, t20n10.90, t20n10.100, t209.80, cit1, cit2, cit3, aar004cp, agr002wp, cshA, gltA, citZ, cit, prpC, cisY, cis, mmgD, citA, gltA1, gltA2, gltA3, cgl0829, prpC1, scd10.20, citA1, citA2, citA3, acly, cg8322, f5e6.2, k7jJ8.14 and citE, where gltA is most preferred.

The enzyme E₁₉ is preferably encoded by genes selected from the group consisting of pckA, pck1, pck2, cg10924, cg17725, cg17725, pckG, ppcK, cgl2863, pck and 2sck36.02.

The enzyme E₂₀ is preferably encoded by genes selected from the group consisting of oadA, oadB, oadC, oadG, oag3, eda, dcoA, oadA1, oadA2, pycB and mmdB.

The enzyme E₂₁ is preferably encoded by genes selected from the group consisting of myn8.7, glt1, adr290wp, gltB, gltD, glt1, gls1, gitA, glt, glxD, gltD1, gltD2, gdh2, agl040Cp, gdhA1, gdhA, gdhA2, gluD, gluD1, gluD2, rocG, ypcA, gudB, t11i18.2, t2i1.150, mrg7.13, f19c24.7, gdh, gdh1, gdh2, gdh3, got1, got2, cg4233, cg8430, f23n19.17, f13j11.16, t26c19.9, f7f1.18, F10N7.200, t1611.170, f15n18.110, t20d1.70, aat1, aat2, ab1038wp, afr211cp, agx1, bna4, aatA, aatB, ybdL, aspC, yfbQ, aat, avtA1, avtA2, tyrB, avtA, avtB, argD1, argD2, aspB1, aspB2, aspB3, aspB, aspC1, aspC2, aspC3, aspC4, RS05143, aspAT, ywfG, yhdR, argD, mtnV, alaT, hisC, avtA1, avtA2, avtA3, cgl0240, cgl1103, cgl2599, cg12844, 2sck36.07c, sc9e12.21, sc2h4.04c, tyrB, gtp, gtp1, gtp2, cg1640, f20d23.34, f26f24.16, f24j13.15, t10d10.20 and agr085wp, where aspC, aatA, gdh, gudB, gdhA, gltB and gltD are especially preferred.

The enzyme E₂₁ is preferably by genes selected from the group consisting of myn8.7, glt1, adr290wp, gltB, gltD, glt1, gls1, gltA, glt, glxD, gltD1, gltD2, gdh2, agl040Cp, gdhA1, gdhA, gdhA2, gluD, gluD1, gluD2, rocG, ypcA,

The enzyme E₂₂ is preferably encoded by genes selected from the group consisting of me, me1, me2, me3, mae, mae1, mae2, sfcA, sfcA1, maeA, maeB, tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7g11.23, malS1, malS2, dme, maeB1, maeB2, mdh, mdh1, mdh2, dmel_cg10120, dmel_cg10120, dmel-cg5889, f19k16.27, f6f22.7, t22p22.60, f18a17.1, mod1, tme, mao, cgl3007, malS and malE.

The enzyme E₂₃ is preferably encoded by genes selected from the group consisting of me, me1, me2, me3, mae, mae1, mae2, sfcA, sfcA1, maeA, maeB, tme, yqkJ, ywkA, yqkJ, malS, ytsJ, mleA, mleS, mez, sce59.10c, 2sc7g11.23, malS1, malS2, dme, maeB1, maeB2, mdh, mdh1, mdh2, dmel_cgl0120, dmelcg10120, dmel-cg5889, f19k16.27, f6f22.7, t22p22.60, f18a17.1, mod1, tme, mao, cgl3007, malS and malE.

Furthermore, it is preferred in accordance with the invention that, in the event where the increased provision of succinyl-coenzyme A in the cell takes place by means of the above-described pathway (oxaloacetate malate→fumarate→succinyl-coenzyme A), the provision of reduction equivalents in the cell is also increased in a targeted manner.

One possibility of increasing the reduction equivalents consists in increasing the oxidative pentose phosphate pathway. In this context, it is especially preferred that the activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and/or of 6-phosphogluconate dehydrogenase (EC 1.1.1.44), which is preferably encoded by the gnd gene, are increased while, if appropriate, simultaneously inhibiting glycolysis, for example by lowering the activity of glucose 6-phosphate isomerase, as described in WO-A-01/07626. In addition to, or instead of, the directed promotion of the pentose phosphate pathway, it may furthermore be preferred to provide reduction equivalents by supplying, to the cells, ethanol as the carbon source and by promoting, in the cells, the conversion of the ethanol into acetaldehyde by means of alcohol dehydrogenases (EC 1.1.1.1, EC 1.1.1.2, EC 1.1.1.71 or EC 1.1.99.8) and the further conversion of the acetaldehyde into acetyl coenzyme A by means of acetaldehyde dehydrogenases (EC 1.2.1.10). Again, suitable genes for alcohol dehydrogenases and acetaldehyde dehydrogenases, can be found in gene databases which are known to the skilled worker, such as, for example, the KEGG database, the NCBI database or the EMBL database.

A second pathway from oxaloacetate to succinyl-coenzyme A leads via citrate as intermediate. In this case, it is preferred in accordance with a second special embodiment of the above-described first, second or third alternative of the cell according to the invention that the cell, if appropriate in addition to an increased activity of the enzyme E₁₀ or E₁₁, features an activity of at least one of the following enzymes E₁₃ to E₁₆ and E₂₄ to E₂₆ which is increased in comparison with its wild type (see FIG. 8):

-   -   of an enzyme E₁₆, which catalyzes the conversion of oxaloacetate         into citrate;     -   of an enzyme E₂₄, which catalyzes the conversion of citrate into         isocitrate;     -   of an enzyme E₂₅, which catalyzes the conversion of isocitrate         into glyoxalate and succinate;     -   of an enzyme E₂₆, which catalyzes the conversion of glyoxalate         into malate;     -   of an enzyme E₁₃, which catalyzes the conversion of malate into         fumarate;     -   of an enzyme E₁₄, which catalyzes the conversion of fumarate         into succinate;     -   of an enzyme E₁₅, which catalyzes the conversion of succinate         into succinyl-coenzyme A.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₁₃, E₁₄, E₁₅, E₁₅, E₂₄, E₂₅, E₂₆, E₁₃E₁₄, E₁₃E₁₅, E₁₃E₁₆, E₁₃E₂₄, E₁₃E₂₅, E₁₃E₂₆, E₁₄E₁₅, E₁₄E₁₆, E₁₄E₂₄, E₁₄E₂₅, E₁₄E₂₆, E₁₅E₁₆, E₁₅E₂₄, E₁₅E₂₅, E₁₅E₂₆ and E₁₃E₁₄E₁₅E₁₆E₂₄E₂₅E₂₆, where E₁₃E₁₄E₁₅E₁₆E₂₄E₂₅E₂₆ is most preferred.

In this context, it is especially preferred that the enzyme

-   E₁₃ is a fumarate hydratase (EC 4.2.1.2), -   E₁₄ is a succinate dehydrogenase (EC 1.3.99.1 or EC 1.3.5.1) or a     succinate quinone oxidoreductase (1.3.5.1), -   E₁₅ is a succinate coenzyme A ligase (EC 6.2.1.4 or EC 6.2.1.5), -   E₁₆ is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8), -   E₂₄ is an aconitate hydratase (EC 4.2.1.3) and -   E₂₅ is an isocitrate lyase (EC 4.1.3.1) and -   E₂₆ is a malate synthase (EC 2.3.3.9).

Preferred genes for the enzymes E₁₃ to E₁₆ are those which have already been described above in connection with the first pathway from oxaloacetate to succinyl-coenzyme A.

The enzyme E₂₄ is preferably encoded by genes selected from the group consisting of aco1, aco2, ratireb, dmel-CG4706, dmel-CG4900, dmel-cg6342, cg9244, t3p4.5, f10m23.310, f4b14.100, ad1032Wp, afr629wp, acnA, acnB, acnC, acnD, rpfA, acnA1, acnA2, acnM, citB, leuC, cg11540, sacA, can and aco, where acnA and acnB are especially preferred.

The enzyme E₂₅ is preferably encoded by genes selected from the group consisting of msd21.4, icl1, icl2, ad1066cp, agl057wp, aceA, icl, aceAa, aceAb, cgl0097 and cgl2331, where aceA is especially preferred. In accordance with a particular embodiment, genes which are preferred are those which code for an isocitrate lyase which is deregulated at the gene level or protein level.

The enzyme E₂₆ is preferably encoded by genes selected from the group consisting of med24.5, mlsS1, acr268cp, masA, glcB, aceB, mls, glcB-1, glcB-2, cgl2329, masZ, aceB1, aceB2 and mas, where the aceB gene is especially preferred.

Again, the nucleotide sequences of suitable genes of the enzymes E₂₄ to E₂₆ can be found in the KEGG database, the NCBI database or the EMBL database.

When the provision of oxaloacetate from phosphoenolpyruvate or from pyruvate is promoted by increasing the activity of the enzyme E₁₀ or E₁₁, the succinate which is formed, besides glyoxalate, upon cleavage of the isocitrate by the isocitrate lyase may also be utilized for the formation of succinyl-coenzyme A. Furthermore, it may be advantageous in this second pathway from the oxaloacetate to the succinate to reduce the activity of an enzyme E₂₇, which catalyzes the conversion of isocitrate into 2-oxoglutarate and which preferably takes the form of an isocitrate dehydrogenase (EC 1.1.1.41 or EC 1.1.1.42). Preferably, the isocitrate dehydrogenase takes the form of an enzyme which is encoded by a gene selected from the group consisting of idh1, idh2, cg7176, cg7176, cg7176, f20d21.16, f12p19.10, t15n1.80, idp1, idp2, idp3, aal022Wp, aer061Cp, idhC, idhM, icdA, icd, idh, icd1, icd2, leuB, citC, citC, cgl0664, leuB2, idh3A, idg3B, idh3G, cg12233, dmel-CG5028, dmel-CG6439, f6p23.14, f23e12.180, f8d20.160, f12e4.20, ad1223wp and afrl37cp, where icdA and citC are especially preferred.

A third pathway from the oxaloacetate to the succinyl-coenzyme A leads via 2-oxoglutarate as intermediate. In this case, it is preferred in accordance with a third special embodiment of the above-described first, second or third alternative of the cell according to the invention that the cell features an activity of at least one of the following enzymes E₁₆, E₂₄, E₂₇ and E₂₈ which is increased in comparison with its wild type, if appropriate in addition to an increased activity of the enzyme E₁₀ or E₁₁ (see FIG. 9):

-   -   of an enzyme E₁₆, which catalyzes the conversion of oxaloacetate         into citrate;     -   of an enzyme E₂₄, which catalyzes the conversion of citrate into         isocitrate;     -   of an enzyme E₂₇, which catalyzes the conversion of isocitrate         into 2-oxoglutarate;     -   of an enzyme E₂₈, which catalyzes the conversion of         2-oxoglutarate into succinyl-coenzyme A.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₁₅, E₂₄, E₂₇, E₂₈, E₁₆E₂₄, E₁₆E₂₇, E₁₆E₂₈, E₂₄E₂₇, E₂₄E₂₈, E₂₇E₂₈, E₁₆E₂₄E₂₇, E₁₆E₂₄E₂₈, E₂₄E₂₇E₂₈ and E₁₆E₂₄E₂₇E₂₈, where E₁₆E₂₄E₂₇E₂₈ is most preferred.

In this context, it is especially preferred that the enzyme

-   E₁₆ is a citrate synthase (EC 2.3.3.1 or EC 2.3.3.8), -   E₂₄ is an aconitate hydratase (EC 4.2.1.3), -   E₂₇ is an isocitrate dehydrogenase (EC 1.1.1.41 or EC 1.1.1.42) and -   E₂₈ is a 2-oxoglutarate synthase (EC 1.2.7.3).

Preferred genes for the enzymes E₁₆, E₂₄ and E₂₇ are those which have already been described above in connection with the first and second pathway from the oxaloacetate to the succinyl-coenzyme A.

The enzyme E₂₈ is preferably encoded by genes selected from the group consisting of korA, korB, kor D, korA1, korA2, korB1, korB2, oorA, oorB, oorC, oorD, oforA, oforB, porA, porB, porA1, porA2, porA3, porA4, porG, porA1, porG2, porB1, porB2, porB3, SCD20.12c, SCD20.13c, SCAH10.34c, SCAH10.35c, korG, orA, orB, korA1 and korG2. Furthermore, E₂₈ may also take the form of a dehydrogenase complex consisting of a plurality of subunits which have different enzymatic activities. In particular, it may take the form of a dehydrogenase complex comprising an oxoglutarate dehydrogenase (EC 1.2.4.2), a dihydrolipoyl dehydrogenase (EC 1.8.1.4) and a dihydrolipoyllysine-residue succinyl transferase (EC 2.3.1.61). In this context, the oxoglutarate dehydrogenase (EC 1.2.4.2) is preferably encoded by genes selected from the group consisting of ogdh, ghdhl, loc239017, mgc68800, mgc80496, cg11661, t22e16.70, mpA24.10, kgd1, aer374cp, sucA, odhA, kgdA and cgl1129, where sucA and odhA are especially preferred. The dihydrolipoyl dehydrogenase (EC 1.8.1.4) is preferably encoded by genes selected from the group consisting of dld, dld-prov, dldh, cg7430, t2j15.6, k14a17.6, at3g17240, mgd8.71pd1, afr512wp, dld1, 1pd, tb03.26j7.650, tb04.3m17.450, tb927.8.7380, tb08.10k10.200, lpdA, lpdG, lpdV, lpd3, acoD, IpdA1, lpdA2, lpdA3, odhL, pdhD, pdhD1, pdhD2, pdhD3, pdhD42, lpdAch1, lpdAch2, lpdAc, acoL, bfmbC, bkdD, cgl0366, cg10688, scm1.17c, pdhL, sck13.11, 1pdB2 and dld1, where lpd is especially preferred. In this context, the dihydrolipoyllysine-residue succinyl transferase (EC 2.3.1.61) is preferably encoded by genes selected from the group consisting of dlst, dlst-prov, mgc89125, dmel_CG5214, f10m23.250, k13p22.8, kgd2agl200wp, kgd2, odhB, sucB, aceF, kgdB, sucB1, sucB2, pdhC, dlaT, kgd, sc5F7.20 and sc4B10.24c, where sucB and odhB are especially preferred.

The nucleotide sequences of suitable genes of the enzyme E₂₈ or of the abovementioned subunits of the enzyme E₂₈, can, again, be found in the KEGG database, the NCBI datase or the EMBL database.

The above-described pathways from the oxaloacetate to the succinyl-coenzyme A depart from phosphoenolpyruvate or from pyruvate as substrate precursors. In this context, it may furthermore be preferred to genetically modify the cells in such a way that they are capable of providing especially large amounts of pyruvate or phosphoenolpyruvate starting from carbohydrates and/or from glycerol.

In the event that the cells are capable of utilizing glycerol as nutrient source, it is preferred that the cell according to the invention displays an activity of at least one, preferably all, of the following enzymes E₂₉ to E₄₂ which is increased in comparison with its wild type:

-   -   of an enzyme E₂₉, which facilitates the diffusion of glycerol         into the cell,     -   of an enzyme E₃₀, which catalyzes the conversion of glycerol         into glycerol 3-phosphate, of an enzyme E₃₁, which catalyzes the         conversion of glycerol 3-phosphate into dihydroxyacetone         phosphate,     -   of an enzyme E₃₂, which catalyzes the transfer of sulfur to the         sulfur acceptor thioredoxin 1,     -   of an enzyme E₃₃, which catalyzes the hydrolysis of         phospholipids with formation of alcohols and glycerol,     -   of an enzyme E₃₄, which catalyzes the transport of glycerol         3-phosphate into the cell in exchange for phosphate;     -   of an enzyme E₃₅, which catalyzes the conversion of         dihydroxyacetone phosphate into glyceraldehyde 3-phosphate,     -   of an enzyme E₃₆, which catalyzes the conversion of         glyceraldehyde 3-phosphate into 1,3-biphosphoglycerate,     -   of an enzyme E₃₇, which catalyzes the conversion of         1,3-biphosphoglycerate into 3-phosphoglycerate,     -   of an enzyme E₃₈, which catalyzes the conversion of         3-phosphoglycerate into 2-phosphoglycerate,     -   of an enzyme E₃₉, which catalyzes the conversion of         2-phosphoglycerate into phosphoenolpyruvate,     -   of an enzyme E₄₀, which catalyzes the conversion of         phosphoenolpyruvate into pyruvate,     -   of an enzyme E₄₁, which catalyzes the conversion of glycerol         into dihydroxyacetone,     -   of an enzyme E₄₂, which catalyzes the conversion of         dihydroxyacetone into dihydroxyacetone phosphate.

In this context, cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is reduced: E₂₉, E₃₀, E₃₁, E₃₂, E₃₃, E₃₄, E₃₅, E₃₆, E₃₇, E₃₈, E₃₉, E₄₀, E₄₁, E₄₂, E₂₉E₃₀, E₂₉E₃₁, E₂₉E₃₂, E₂₉E₃₃, E₂₉E₃₄, E₂₉E₃₅, E₂₉E₃₆, E₂₉E₃₇, E₂₉E₃₈, E₂₉E₃₉, E₂₉E₄₀, E₂₉E₄₁, E₂₉E₄₂, E₃₀E₃₁, E₃₀E₃₂, E₃₀E₃₃, E₃₀E₃₄, E₃₀E₃₅, E₃₀E₃₆, E₃₀E₃₇, E₃₀E₃₈, E₃₀E₃₉, E₃₀E₄₀, E₃₀E₄₁, E₃₀E₄₂, E₃₁E₃₂, E₃₁E₃₃, E₃₁E₃₄, E₃₁E₃₅, E₃₁E₃₆, E₃₁E₃₇, E₃₁E₃₈, E₃₁E₃₉, E₃₁E₄₀, E₃₁E₄₁, E₃₁E₄₂, E₃₂E₃₃, E₃₂E₃₄, E₃₂E₃₅ E₃₂E₃₆, E₃₂E₃₇, E₃₂E₃₈, E₃₂E₃₉, E₃₂E₄₀, E₃₂E₄₁, E₃₂E₄₂, E₃₃E₃₄, E₃₃E₃₅, E₃₃E₃₆, E₃₃E₃₇, E₃₃E₃₈, E₃₃E₃₉, E₃₃E₄₀, E₃₄E₄₁, E₃₃E₄₂, E₃₄E₃₅, E₃₄E₃₆, E₃₄E₄₇, E₃₄E₃₈, E₃₄E₃₉, E₃₄E₄₀, E₃₄E₄₁, E₃₄E₄₂, E₃₅E₃₆, E₃₅E₃₇, E₃₅E₃₈, E₃₅E₃₉, E₃₅E₄₀, E₃₅E₄₁, E₃₅E₄₂, E₃₆E₃₇, E₃₆E₃₈, E₃₆E₃₉, E₃₆E₄₀, E₃₆E₄₁, E₃₆E₄₂, E₃₇E₃₈, E₃₇E₃₉, E₃₇E₄₀, E₃₇E₄₁, E₃₇E₄₂, E₃₈E₃₉, E₃₉E₄₀, E₃₉E₄₁, E₃₉E₄₂, E₄₀E₄₁, E₄₀E₄₂, E₄₁E₄₂ and E₂₉E₃₀E₃₁E₃₂E₃₃E₃₄E₃₅E₃₆E₃₇E₃₈E₃₉-E₄₀E₄₁E₄₂.

In this context, it is especially preferred that the enzyme

-   E₂₉ is an aquaglyceroporin (glycerol facilitator) which is     preferably encoded by the glpF gene, -   E₃₀ is a glycerol kinase (EC 2.7.1.30) which is preferably encoded     by the glpK gene, -   E₃₁ is a glycerol 3-phosphate dehydrogenase (EC 1.1.99.5),     preferably an FAD-dependent glycerol 3-phosphate dehydrogenase,     where the glycerol 3-phosphate dehydrogenase is preferably encoded     by the gipA gene, the glpB gene, the glpC gene or the glpD gene,     especially preferably by the glpD gene, -   E₃₂ is a sulfur transferase which is encoded by the glpE gene, -   E₃₃ is a glycerol phosphodiesterase (EC 3.1.4.46) which is     preferably encoded by the glpQ gene, -   E₃₄ is a glycerol 3-phosphate permease which is preferably encoded     by the glpT gene, -   E₃₅ is a triose phosphate isomerase (EC 5.3.1.1), -   E₃₆ is a glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), -   E₃₇ is a phosphoglycerate kinase (EC 2.7.2.3), -   E₃₈ is a phosphoglycerate mutase (EC 5.4.2.1), -   E₃₉ is an enolase (EC 4.2.1.11), -   E₄₀ is a pyruvate kinase (EC 2.7.1.40), -   E₄₁ is a glycerol dehydrogenase (EC 1.1.1.6) which is preferably     encoded by the gldA gene, and -   E₄₂ is a dihydroxyacetone kinase (EC 2.7.1.29) which is preferably     encoded by the dhaK gene.

The gene sequences of the abovementioned enzymes can, again, be found in the gene databases which are known to the skilled worker, in particular the KEGG database, the NCBI database or the EMBL database.

Furthermore, the gap gene, which codes for glyceraldehyde 3-phosphate dehydrogenase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), the tpi gene, which codes for triose phosphate isomerase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), and the pgk gene, which codes for 3-phosphoglycerate kinase (Eikmanns (1992), Journal of Bacteriology 174: 6076-6086), are also known from other sources.

Using the known genes of the enzymes E₂₉ to E₄₂, it is possible to prepare genetically modified cells in which at least one, preferably at least two, more preferably at least three and most preferably all activities of the enzymes E₂₉ to E₄₂ has been increased by means of the techniques (mutation of the enzyme or increase in the expression of the enzyme) described at the outset in connection with the enzyme E₁. These cells are capable of being cultured in the presence of glycerol as the only carbon source (or else together with carbohydrates as further carbon source).

In addition to increasing one or more of the enzymatic activities E₂₉ to E₄₂, it may, in the event that the cell is capable of utilizing glycerol as carbon source, also be advantageous when the following genes are expressed, preferably heterologously expressed, in the cells according to the invention:

-   -   the glpG gene or the 3925 gene,     -   the glpX gene,     -   the dhaR gene, the ycgU gene or the b1201 gene,     -   the fsa gene, the mipB gene, the ybiZ gene or the B0825 gene,     -   the talC gene, the fsaB gene, the yijG gene or the b3946 gene.

Again, the nucleotide sequences of these genes can be found in the KEGG database, the NCBI database or the EMBL database.

In the event that the cells are capable of utilizing carbohydrates as nutrient source, it is preferred that the cell according to the invention features an activity of at least one, preferably of all, of the following enzymes E₄₃ to E₄₅ and E₃₆ to E₄₀ which is increased in comparison with its wild type:

-   -   of an enzyme E₄₃, which catalyzes the conversion of α-D-glucose         6-phosphate into β-D-fructose 6-phosphate, of an enzyme E₄₄,         which catalyzes the conversion of β-D-fructose 6-phosphate into         β-D-fructose 1,6-biphosphate,     -   of an enzyme E₄₅, which catalyzes the conversion of

β-D-fructose 1,6-biphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate,

-   -   of an enzyme E₃₆, which catalyzes the conversion of         glyceraldehyde 3-phosphate into 1,3-biphosphoglycerate,     -   of an enzyme E₃₇, which catalyzes the conversion of         1,3-biphosphoglycerate into 3-phosphoglycerate,

-   of an enzyme E₃₈, which catalyzes the conversion of     3-phosphoglycerate into 2-phosphoglycerate,     -   of an enzyme E₃₉, which catalyzes the conversion of         2-phosphoglycerate into phosphoenolpyruvate, and     -   of an enzyme E₄₀, which catalyzes the conversion of         phosphoenolpyruvate into pyruvate.

In this context genetically modified cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₃₆, E₃₇, E₃₈, E₃₉, E₄₀, E₄₃, E₄₄, E₄₅, E₃₆E₃₇, E₃₆E₃₈, E₃₆E₃₉, E₃₆E₄₀, E₃₆E₄₃, E₃₆E₄₄, E₃₆E₄₅, E₃₇E₃₈, E₃₇E₃₉, E₃₇E₄₀ E₃₇E₄₃, E₃₇E₄₄, E₃₇E₄₅, E₃₈E₃₉, E₃₈E₄₀, E₃₈E₄₃, E₃₈E₄₄, E₃₈E₄₅, E₃₉E₄₀ E₃₉E₄₃, E₃₉E₄₄, E₃₉E₄₅, E₄₀E₄₃, E₄₀E₄₄, E₄₀E₄₅, E₄₃E₉₄, E₄₃E₄₅, E₄₄E₄₅ and E₃₆E₃₇E₃₈E₃₉E₄₀E₄₃E₄₄E₄₅

In this context, it is especially preferred that the enzyme

-   E₄₃ is a glucose 6-phosphate isomerase (EC 5.3.1.9), -   E₄₄ is a 6-phosphofructo kinase (EC 2.7.1.11), -   E₄₅ is a fructose bisphosphate aldolase (EC 4.1.2.13), -   E₃₆ is a glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12), -   E₃₇ is a phosphoglycerate kinase (EC 2.7.2.3), -   E₃₈ is a phosphoglycerate mutase (EC 5.4.2.1), -   E₃₉ is an enolase (EC 4.2.1.11) and -   E₄₀ is a pyruvate kinase (EC 2.7.1.40).

Again, the nucleotide sequences of these genes can be found be found in the KEGG database, the NCBI database or the EMBL database.

In the event that the cell is capable of utilizing carbohydrates as carbon source, it is furthermore preferred to promote not only the abovementioned enzymes E₄₃ to E₄₅ and E₃₆ to E₄₀, but also the uptake of glucose into the cells, for example by increasing the activity of enzymes of the phosphotransferase system, in particular those enzymes which are encoded by ptsI, ptsH and ptsM genes, or by enhancing glucokinase (EC 2.7.1.2), which is preferably encoded by the glk gene. In this context, reference is made in particular to U.S. Pat. No. 6,680,187, U.S. Pat. No. 6,818,432, U.S. Pat. No. 6,913,910 and U.S. Pat. No. 6,884,614, whose disclosure content with regard to the possibilities for overexpressing the ptsI, ptsH, ptsM and glk genes is hereby incorporated by reference and forms part of the disclosure of the present invention. In the event that carbohydrates act as carbon source, it may also be advantageous to promote the pentose phosphate pathway in a targeted manner, for example by increasing the activity of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and of 6-phosphogluconate dehydrogenase (EC 1.1.1.44), which is preferably encoded by the gnd gene, while, if appropriate, simultaneously inhibiting glycolysis, for example by weakening the activity of glucose 6-phosphate isomerase, as is described in WO-A-01/07626.

In the event that, according to the special embodiment of the cell according to the invention where methylmalonate semialdehyde is formed as precursor and succinyl-coenzyme A as intermediate, the cells form 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via oxaloacetate and pyruvate as intermediates, it may furthermore be preferred to reduce the activity of at least one, preferably of all, of the following enzymatic activities in the cell:

-   -   of an enzyme which catalyzes the conversion of oxaloacetate into         phosphoenolpyruvate, such as, for example, phosphoenylpyruvate         carboxykinase (EC 4.1.1.49) (see also DE-A-199 50 409),     -   of an enzyme which catalyzes the conversion of pyruvate into         acetate such as, for example, pyruvate oxidase (EC 1.2.2.2) (see         also DE-A-199 51 975),     -   of an enzyme which catalyzes the conversion of α-D-glucose         6-phosphate into βD-fructose 6-phosphate (see also U.S. Ser. No.         09/396,478),     -   of an enzyme which catalyzes the conversion of pyruvate into         lactate such as, for example, 1-lactate dehydrogenase (EC         1.1.1.27) or lactate-malate transhydrogenase (EC 1.1.99.7),     -   of an enzyme which catalyzes the conversion of pyruvate into         acetyl-coenzyme A such as, for example, pyruvate dehydrogenase         (EC 1.2.1.51),     -   of an enzyme which catalyzes the conversion of pyruvate into         acetyl phosphate such as, for example, pyruvate oxidase (EC         1.2.3.3),     -   of an enzyme which catalyzes the conversion of pyruvate into         acetate, such as, for example, pyruvate dehydrogenase (EC         1.2.2.2),     -   of an enzyme which catalyzes the conversion of pyruvate into         phosphoenolpyruvate such as, for example, phosphoenolpyruvate         synthase (EC 2.7.9.2) or pyruvate, phosphate dikinase (EC         2.7.9.1),     -   of an enzyme which catalyzes the conversion of pyruvate into         alanine such as, for example, alanine transaminase (2.6.1.2) or         alanine-oxo-acid transaminase (EC 2.6.1.12), and/or     -   of an enzyme which converts pyruvate into acetolactate such as,         for example, acetohydroxy acid synthase (EC 2.2.1.6).

Cells which are especially preferred in accordance with the invention and which are capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxybutyric acid from carbohydrates as carbon source via succinyl-coenzyme A as intermediate and in which one or more of the abovementioned enzymatic activities, in particular one of the enzymatic activities E₁ to E₄₅, more preferably the enzymatic activities E₁, E₁E₂E₃E₄, E₁E₄E₅E₆E₇ or and E₁E₄E₅E₇, can be increased are those microorganisms which have been described by Bennett et al., Metab. Eng. (2005), 7 (3), pages 229 to 239, Bennett et al., Biotechnol. Bioeng. (2005), 90 (6), pages 775 to 779, Bennett et al., Biotechnol. Prog. (2005), 21 (2), pages 358 to 365, Bennett et al. (2005), Appl. Microbiol. Biotechnol., 67 (4), pages 515 to 523, Vemuri et al. (2002), Applied and Environmental Microbiology 68 (4), pages 1715 to 1727 and in U.S. Pat. No. 6,455,284.

If, according to the first special embodiment of the cell according to the invention, the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoates based on 3-hydroxyisobutyric acid starting from L-glutamate as carbon source takes place via succinyl-coenzyme A as intermediate, it is, in a further special embodiment of the cell according to the invention, in which methylmalonat semialdehyde is formed as precursor and succinyl-coenzyme A as intermediate, furthermore preferred in accordance with the invention that it features an activity of at least one of the, preferably of the two, following enzymes E₂₈ and E₄₆ which is increased in comparison with its wild type (see FIG. 10):

-   -   of an enzyme E₄₆, which catalyzes the conversion of L-glutamate         into 2-oxoglutarate;     -   of an enzyme E₂₈, which catalyzes the conversion of         2-oxoglutarate into succinyl-coenzyme A.

In this context, it is especially preferred that the enzyme

-   E₄₆ is a glutamate synthase (EC 1.4.1.13 or EC 1.4.1.14), a     glutamate dehydrogenase (EC 1.4.1.2, EC 1.4.1.3 or EC 1.4.1.4) or an     aspartate transaminase (EC 2.6.1.1 or EC 2.6.1.2) and -   E₂₈ is a 2-oxoglutarate synthase (EC 1.2.7.3).

Preferred as enzyme E₂₈ are those which have already been mentioned at the outset as preferred enzymes E₂₈.

The enzyme E₄₆ is preferably encoded by the genes selected from the group consisting of: myn8, glt1, adr290wp, gltB, gltD, yeiT, aegA, ygfT, gltD-1, gltD-2, glt1, glt2, gls1, gltA, glt, glxD, gltA, yerD, cgl0184, cgl0185, sc3c9.12, gdh1, gdh2, agl40cp, gdhA, gdhA1, gdhA2, gluD, rocG, ypcA, gudB, gluD, gdhA, gdhA2, gdh, gdhA-1, gdhA2-2, gdhA-3, gluD1, gluD2, glud1-prov, glud1a, t11I18.2, t2I1.150, mrg7.13, got1, got2, caspat, got2-prov, xr406-prov, 406-prov, cg4233, cg4233, cg8430, cg8430, f23n19.17, f13j11.16, t26c19.9, f7f1.18, f10n7.200, t1611.170, f15n18.110, t20d1.70, aat, aat1, aat2, ab1038wp, afr211cp, agx1, bnA4, aatA, aatB, ybdL, aspC, yfbQ, ydcR, avtA2, aspC-1, aspC-2, aspC-3, aspC-4, aspB, aspB-1, aspB-2, aspB-3, aspB-4, argD1, argD2, aatAc, ywfG, mtnV, alaT, avtA1, avtA2, avtA3, cgl0240, cgl1103, cgl2599, cgl2844, dapC, 2sck36.07c, sc9e12.21, sc2h4.04c, aspB1, aspB2, aspB3, tyrB, gpt, gpt1, gpt2, mgc82097, cgl640, c32f10.8, f20d23.34, f26f24.16, f24j13.15, t10d10.20 or agrwp.

Again, the nucleotide sequences of these genes can be found be found in the KEGG database, the NCBI database or the EMBL database.

In accordance with a second special embodiment of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate semialdehyde as precursor, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place via propionyl-coenzyme A as intermediate, where the cell is capable of preferentially utilizing carbohydrates, glycerol, methane or methanol as carbon source. In this context, a variety of pathways exist for arriving at 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, departing from propionyl-coenzyme A.

In accordance with a first alternative of this second special embodiment of the cell according to the invention, the formation of intermediate propionyl-coenzyme A takes place via acetyl-coenzyme A as further intermediate. In this context, it is especially preferred that the cell features an activity of at least one of the following enzymes E₄, E₅ and E₄₇ to E₅₂ which is increased in comparison with its wild type (see FIG. 11):

-   -   of an enzyme E₄₇, which catalyzes the conversion of         acetyl-coenzyme A into malonyl-coenzyme A;     -   of an enzyme E₄₈, which catalyzes the conversion of         malonyl-coenzyme A into malonate semialdehyde;     -   of an enzyme E₄₉, which catalyzes the conversion of malonate         semialdehyde into 3-hydroxypropionate;     -   of an enzyme E₅₀, which catalyzes the conversion of         3-hydroxypropionate into 3-hydroxypropionyl-coenzyme A;     -   of an enzyme E₅₁, which catalyzes the conversion of         3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;     -   of an enzyme E₅₂, which catalyzes the conversion of         acryloyl-coenzyme A into propionyl-coenzyme A;     -   of an enzyme E₅, which catalyzes the conversion of         propionyl-coenzyme A into methylmalonate semialdehyde;     -   of an enzyme E₄, which catalyzes the conversion of         methylmalonate semialdehyde into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₄₇, E₄₈, E₄₉, E₅₀, E₅₁, E₅₂, E₄, E₅ and E₄₇E₄₈E₄₉E₅₀E₅₁E₅₂E₄E₅.

Furthermore, it is particularly preferred in this context that the enzyme

-   E₄ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a     3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35), -   E₅ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), -   E₄₇ is a malonyl-coenzyme A decarboxylase (EC 4.1.1.9), a     malonate-coenzyme A transferase (EC 2.8.3.3), a     methylmalonyl-coenzyme A carboxy-transferase (EC 2.1.3.1) or an     acetyl-coenzyme A carboxylase (EC 6.4.1.2), -   E₄₈ is a malonate-semialdehyde dehydrogenase (EC 1.2.1.18), -   E₄₉ is a 3-hydroxypropionate dehydrogenase (EC 1.1.1.59), -   E₅₀ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), -   E₅₁ is an enoyl-coenzyme A hydratase (EC 4.2.1.17) and -   E₅₂ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3).

Preferred genes for the enzymes E₄ and E₅ are those which have already been described above in connection with the first special embodiment of the cell according to the invention.

The enzyme E₄₇ is preferably encoded by genes selected from the group consisting of mlycd, t19 b17.4, tb08.2904.110, matA, acac, acaca, acacb, f5j5.21, f15c21.2, t8p21.5, acc1, aar071wp, accA, accB, accC, accD, accC1, accC2, mmdA, fabG, accD1, accD2, accD3, cgl0831, accBC, dtsR1, accDA, scc24.16c and cgl1327, where accA, accC and accD are most preferred.

The enzyme E₄₈ is preferably encoded by the iolD gene.

The enzyme E₅₁ is preferably encoded by genes selected from the group consisting of echS1, ehhadh, hadha, echs1-prov, cg4389, cg4389, cg6543, cg6984, cg8778, ech-1, ech-2, ech-3, ech-4, ech-5, ech-6, ech-7, FCAALL.314, fcaall.21, fox2, eci1, eci2, paaF, paaG, yfcX, fadB, faoA, rpfF, phaA, phaB, echA1, echA2, echA3, echA4, echA5, echA6, echA7, echA8, echA9, echA9, echA10, echA11, echA12, echA13, echA14, echA15, echA16, echA17, echA18, echA19, echA20, echA21, fad-1, fad-2, fad-3, dcaE, hcaA, fadJ, rsp0671, rsp0035, rsp0648, rsp0647, rs03234, rs03271, rs04421, rs04419, rs02820, rs02946, paaG1, paaG2, paaG3, ech, pksH, ydbS, eccH1, eccH2, pimF, fabJ1, fabJ2, caiD2, ysiB, yngF, yusL, fucA, cgl0919, scf41.23, scd10.16, sck13.22, scp8.07c, stbac16h6.14, sc5f2a.15, sc6a5.38, hbd-1, hbd-2, hdb-3, hdb-4, hdb-5, hdb-6, hdb-7, hdb-8, hdb-9, hdb-10, fad-1, fad-2, fad-3, fad-4, fad-5, paaF-1, paaF-2, paaF-3, paaF-4, paaF-5, paaF-6, paaF-7 and crt.

The enzyme E₅₂ is preferably encoded by genes selected from the group consisting of acadl, acadm, acad10, acad11, acadm-prov, acadl-prov, mgc81873, cg12262, cg4703, cg4860, f3e22.5, af1213wp, acdC, fadE13, acd-1, acd-2, acd-3, acd-4, acd-5, acd-6, acd-7, acd-8, acd-9, acd-10, acd-11, acd-12, acd, fadE1, fadE2, fadE3, fadE4, fadE5, fadE6, fadE7, fadE13, fadE14, fadE15, fadE16, fadE17, fadE18, fadE19, fadE20, fadE21, fadE22, fadE23, fadE26, fadE27, fadE30, fadE31, fadE33, fadE35, fadE38, fadE45, fadE, caiA, aidB, RSp0036, RS03588, mmgC, acdA-3, bcd, acdA, acdH1, acdH2, acdH3, aidB, acdI and acdH.

The nucleotide sequences of suitable genes for the enzymes E₄₇ to E₅₂, in particular also of the enzymes E₄₉ and E₅₀, can be found in the KEGG database, the NCBI database or the EMBL database.

According to a second alternative of this second special embodiment of the cell according to the invention, the formation of the intermediate propionyl-coenzyme A also takes place via acetyl-coenzyme A as further intermediate, where, according to this alternative, the propionyl-coenzyme A is not converted directly into the methylmalonate semialdehyde, but via methylmalonyl-coenzyme A. In this context, it is especially preferred that the cell features an activity of at least one of the following enzymes E₂ to E₄, E₆, E₇ and E₄₇ to E₅₂ which is increased in comparison with its wild type (see FIG. 12):

-   -   of an enzyme E₄₇, which catalyzes the conversion of         acetyl-coenzyme A into malonyl-coenzyme A;     -   of an enzyme E₄₈, which catalyzes the conversion of         malonyl-coenzyme A into malonate semialdehyde;     -   of an enzyme E₄₉, which catalyzes the conversion of malonate         semialdehyde into 3-hydroxypropionate;     -   of an enzyme E₅₀, which catalyzes the conversion of         3-hydroxypropionate into 3-hydroxypropionyl-coenzyme A;     -   of an enzyme E₅₁, which catalyzes the conversion of         3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;     -   of an enzyme E₅₂, which catalyzes the conversion of         acryloyl-coenzyme A into propionyl-coenzyme A;     -   of an enzyme E₇, which catalyzes the conversion of         propionyl-coenzyme A into (S)-methylmalonyl-coenzyme A;     -   of an enzyme E₆, which catalyzes the conversion of         (S)-methylmalonyl-coenzyme A into (R)-methylmalonyl-coenzyme A;     -   of an enzyme E₂, which catalyzes the conversion of         (R)-methylmalonyl-coenzyme A into methyl malonate;     -   of an enzyme E₃, which catalyzes the conversion of methyl         malonate into methylmalonate semialdehyde;     -   of an enzyme E₄, which catalyzes the conversion of         methylmalonate-semialdehyde into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred according to the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₂, E₃, E₄, E₅, E₄₇, E₄₈, E₄₉, E₅₀, E₅₁, E₅₂ and E₂E₃E₄E₆E₇E₄₇E₄₈E₄₉E₅₀E₅₁E₅₂.

Preferred enzymes and genes of these enzymes are those genes and enzymes which have already been mentioned above in connection with the enzymes E₂ to E₄, E₆, E₇ and E₄₇ to E₅₂.

According to a third alternative of this first alternative of the second special embodiment of the cell according to the invention, the formation of the intermediate propionyl-coenzyme A also takes place via acetyl-coenzyme A as further intermediate, where, according to this alternative, the propionyl-coenzyme A is, again, not converted directly into methylmalonate-semialdehyde, but via (R)-methylmalonyl-coenzyme A (and not via (S)-methylmalonyl-coenzyme A). In this context, it is especially preferred that the cell features an activity of at least one of the following enzymes E₂ to E₄, E₇ and E₄₇ to E₅₂ which is increased in comparison with its wild type (see FIG. 13):

-   -   of an enzyme E₄₇, which catalyzes the conversion of         acetyl-coenzyme A into malonyl-coenzyme A;     -   of an enzyme E₄₈, which catalyzes the conversion of         malonyl-coenzyme A into malonate semialdehyde;     -   of an enzyme E₄₉, which catalyzes the conversion of malonate         semialdehyde into 3-hydroxypropionate;     -   of an enzyme E₅₀, which catalyzes the conversion of         3-hydroxypropionate into 3-hydroxypropionyl-coenzyme A;     -   of an enzyme E₅₁, which catalyzes the conversion of         3-hydroxypropionyl-coenzyme A into acryloyl-coenzyme A;     -   of an enzyme E₅₂, which catalyzes the conversion of         acryloyl-coenzyme A into propionyl-coenzyme A;     -   of an enzyme E₇, which catalyzes the conversion of         propionyl-coenzyme A into methylmalonyl-coenzyme A;     -   of an enzyme E₂, which catalyzes the conversion of         methylmalonyl-coenzyme A into methylmalonate;     -   of an enzyme E₃, which catalyzes the conversion of methyl         malonate into methylmalonate-semialdehyde;     -   of an enzyme E₄, which catalyzes the conversion of         methylmalonate-semialdehyde into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred according to the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₂, E₃, E₄, E₇, E₄₇, E₄₈, E₄₉, E₅₀, E₅₁, E₅₂ and E₂E₃E₄E₇E₄₇E₄₈E₄₉E₅₀E₅₁E₅₂.

Preferred enzymes and genes of these enzymes are, again, those genes and enzymes which have already been mentioned above in connection with the enzymes E₂ to E₄, E₇ and E₄₇ to E₅₂.

According to a fourth alternative of the second special embodiment of the cell according to the invention, the formation of the intermediate propionyl-coenzyme A also takes place via acetyl-coenzyme A as further intermediate, where, according to this alternative, acetoacetyl-coenzyme A is formed as intermediate. In this context, it may be preferred that the cell features an activity of at least one of the following enzymes E₈ and E₅₃ to E₆₁ which is increased in comparison with its wild type:

-   -   of an enzyme E₅₃, which catalyzes the conversion of         acetyl-coenzyme A into acetoacetyl-coenzyme A;     -   of an enzyme E₅₄, which catalyzes the conversion of         acetoacetyl-coenzyme A into 3-hydroxybutanoyl-coenzyme A;     -   of an enzyme E₅₅, which catalyzes the conversion of         3-hydroxybutanoyl-coenzyme A into crotonyl-coenzyme A;     -   of an enzyme E₅₆, which catalyzes the conversion of         crotonyl-coenzyme A into butyryl-coenzyme A;     -   of an enzyme E₅₇, which catalyzes the conversion of         butyryl-coenzyme A into ethylmalonyl-coenzyme A;     -   of an enzyme E₅₈, which catalyzes the conversion of         ethylmalonyl-coenzyme A into methylsuccinyl-coenzyme A;     -   of an enzyme E₅₉, which catalyzes the conversion of methyl         succinyl-coenzyme A isobutyryl-coenzyme A;     -   of an enzyme E₆₀, which catalyzes the conversion of         isobutyryl-coenzyme A into methacrylyl-coenzyme A;

of an enzyme E₆₁, which catalyzes the conversion of methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;

-   -   of an enzyme E₈, which catalyzes the conversion of         3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred according to the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₈, E₅₃, E₅₄, E₅₅, E₅₆, E₅₇, E₅₈, E₅₉, E₆₀, E₆₁ and E₈E₅₃E₅₄E₅₅E₅₆E₅₇E₅₈E₅₉E₆₀E₆₁.

This metabolic pathway and the enzymes which play a role in this metabolic pathway are described, for example, in Korotkova et al., Journal of Bacteriology (2002), pages 1750 to 1758.

According to a fifth alternative of the second special embodiment of the cell according to the invention, the formation of the intermediate propionyl-coenzyme A takes place via, again, acetyl-coenzyme A as further intermediate, where, according to this alternative, acetoacetyl-coenzyme A is formed as further intermediate but where, in this case, ethylmalonyl-coenzyme A is formed directly from crotonyl-coenzyme A. In this context, it may be preferred that the cell features an activity of at least one of the following enzymes E₈ and E₅₃ to E₅₆ and E₆₂ to E₆₅ which is increased in comparison with its wild type (see FIG. 14):

-   -   of an enzyme E₅₃, which catalyzes the conversion of two         acetyl-coenzyme A units into acetoacetyl-coenzyme A;     -   of an enzyme E₅₄, which catalyzes the conversion of         acetoacetyl-coenzyme A into 3-hydroxybutyryl-coenzyme A;     -   of an enzyme E₅₅, which catalyzes the conversion of         3-hydroxybutyryl-coenzyme A into crotonyl-coenzyme A;     -   of an enzyme E₅₆, which catalyzes the conversion of         crotonyl-coenzyme A into ethylmalonyl-coenzyme A;     -   of an enzyme E₆₂, which catalyzes the conversion of         ethylmalonyl-coenzyme A into methylsuccinyl-coenzyme A;     -   of an enzyme E₆₃, which catalyzes the conversion of         methylsuccinyl-coenzyme A into mesaconyl-coenzyme A;     -   of an enzyme E₆₄, which catalyzes the conversion of         mesaconyl-coenzyme A into β-methylmalyl-coenzyme A;     -   of an enzyme E₆₅, which catalyzes the conversion of         β-methylmalyl-coenzyme A into glyoxylate and propionyl-coenzyme         A.

Then, from propionyl-coenzyme A can in the above-described manner (increasing the activity of one or more of the enzymes E₇, E₂, E₃ and E₄, increasing the activity of one or more of the enzymes E₇, E₆, E₂, E₃ and E₄, or increasing the activity of one of the, or of both, enzymes E₄ and E₅).

In this context, it is especially preferred that the enzyme

-   E₅₃ is a β-ketothiolase (EC 2.3.1.9), -   E₅₄ is an acetoacetyl-coenzyme A reductase (an EC 1.1.1.36), -   E₅₅ is an enoyl-coenzyme A hydratase (EC 4.2.1.17), -   E₅₆ is a crotonyl-coenzyme A decarboxylase, -   E₆₂ is an ethylmalonyl-coenzyme A mutase (EC 5.4.99.2), -   E₆₃ is a methylsuccinyl-coenzyme A dehydrogenase, -   E₆₄ is a mesaconyl-coenzyme A hydratase, and -   E₆₅ is a β-methylmalyl/L-malyl-coenzyme A lyase.

The enzyme E₅₃ is preferably encoded by genes selected from the group consisting of acat1, acat2, loc484063, loc489421, mgc69098, mgc81403, mgc81256, mgc83664, kat-1, erg10, ygeF, atoB, fadAx, phbA-1, phbA-2, atoB-2, pcaF, pcaF-2, phb-A, bktB, phaA, tioL, thlA, fadA, paaJ, phbAf, pimB, mmgA, yhfS, thl, vraB, thl, mvaC, thiL, paaJ, fadA3, fadA4, fadA5, fadA6, cgl12392, catF, sc8f4.03, thiL1, thiL2, acaB1, acaB2, acaB3, acaB4 or, where acat1, acat2, atoB and phbA and the corresponding gene from Rhodobacter sphaeroides are especially preferred.

The enzyme E₅₄ is preferably encoded by genes selected from the group consisting of phbB, fabG, phbN1, phbB2 or cgl12444, where phbB is especially preferred and the corresponding gene from Rhodobacter sphaeroides is especially preferred.

The enzyme E₅₅ is preferably encoded by genes selected from the group consisting of echS1, ehhadh, hadha, echs1-prov, Das Enzym E₅₅ wird vorzugsweise von Genen ausgewählt aus der Gruppe bestehend aus echS1, ehhadh, hadha, echS1-prov, cg4389, cg4389, cg6543, cg6984, cg8778, ech-1, ech-2, ech-3, ech-4, ech-5, ech-6, ech-7, FCAALL.314, fcaall.21, fox2, eci1, eci2, paaF, paaG, yfcX, fads, faoA, rpfF, phaA, phaB, echA1, echA2, echA3, echA4, echA5, echA6, echA7, echA8, echA9, echA9, echA10, echA11, echA12, echA13, echA14, echA15, echA16, echA17, echA18, echA19, echA20, echA21, fad-1, fad-2, fad-3, dcaE, hcaA, fadJ, rsp0671, rsp0035, rsp0648, rsp0647, rs03234, rs03271, rs04421, rs04419, rs02820, rs02946, paaG1, paaG2, paaG3, ech, pksH, ydbS, eccH1, eccH2, pimF, fabJ1, fabJ2, caiD2, ysiB, yngF, yusL, fucA, cgl0919, scf41.23, scd10.16, sck13.22, scp8.07c, stbac16h6.14, sc5f2a.15, sc6a5.38, hbd-1, hbd-2, hdb-3, hdb-4, hdb-5, hdb-6, hdb-7, hdb-8, hdb-9, hdb-10, fad-1, fad-2, fad-3, fad-4, fad-5, paaF-1, paaF-2, paaF-3, paaF-4, paaF-5, paaF-6, paaF-7 and crt

where the corresponding gene from Rhodobacter sphaeroides is especially preferred.

The enzyme which is preferably employed as enzyme E₅₆ is an enzyme from Rhodobacter sphaeroides which is encoded by the DNA sequence with the SEQ ID No 05 and which has the amino acid sequence as shown in SEQ ID No 06.

Suitable genes for the enzyme E₆₂ are selected from the group consisting of mut, mutA, mutB, sbm, sbmA, sbmB, sbm5, bhbA, mcmA, mcmA1, mcmA2, mcmB, mcm1, mcm2, mcm3, icmA, meaA1 and meaA2, where, again, the corresponding gene from Rhodobacter sphaeroides is especially preferred.

Preferred genes for the enzymes E₆₃, E₆₄ and E₆₅ are, in particular, the genes for these enzymes from Rhodobacter sphaeroides.

Further examples of nucleotide sequences of the abovementioned genes can also be found in the KEGG database, the NCBI database or the EMBL database, inter alia.

As has already been explained above, the first alternative of the second preferred embodiment cell according to the invention generates 3-hydroxyisobutyric acid or the polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via propionyl-coenzyme A and acetyl-coenzyme A as intermediates. In this context, it may be meaningful, in principle, to influence not only one or more of the abovementioned enzymatic activities E₂ to E₈ and E₄₇ to E₆₅, but also those enzymatic activities which bring about an increase in the formation of acetyl-coenzyme A in the cell.

In the event that 3-hydroxyisobutyric acid is formed from carbohydrates or glycerol as carbon source, it may be preferred that the cell features an increased activity in an enzyme E₆₆, which catalyzes the conversion of pyruvate into acetyl-coenzyme A. This enzyme E₆₆ preferably takes the form of a pyruvate dehydrogenase (EC 1.2.1.51).

In the event that 3-hydroxyisobutyric acid is formed from C₁-carbon sources such as, for example, methane or methanol, it may be preferred that the cell features an activity of at least one of the enzymes E₆₇ to E₇₁ which is increased in comparison with its wild type:

-   -   of an enzyme E₆₇, which catalyzes the conversion of methane into         methanol;     -   of an enzyme E₆₈, which catalyzes the conversion of methanol         into formaldehyde;     -   of an enzyme E₆₉, which catalyzes the conversion of formaldehyde         into 5,10-methylenetetrahydrofolate;     -   of an enzyme E₇₀, which catalyzes the conversion of         5,10-methylenetetrahydrofolate into 5-methyltetrahydrofolate;     -   of an enzyme E₇₁, which catalyzes the conversion of         5-methyltetrahydrofolate into acetyl-coenzyme A.

In this context, it is especially preferred that the enzyme

-   E₆₇ is a methane monooxygenase (EC 1.14.13.25), -   E₆₈ is a methanol dehydrogenase (EC 1.1.1.244), -   E₆₉ is a methylmalonate-semialdehyde dehydrogenase (EC 1.2.1.27), -   E₇₀ is a methylenetetrahydrofolate reductase (EC 1.5.1.20), -   E₇₁ is a carbon monoxide dehydrogenase (EC 1.2.99.2).

The nucleotide sequences of suitable genes for the enzymes E₆₃ to E₆₇ can be found in the KEGG database, the NCBI database or the EMBL database.

According to a third special embodiment of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via methylmalonate-semialdehyde as precursor, it is preferred that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place via acryloyl-coenzyme A as intermediate, where the cell is capable of preferentially utilizing carbohydrates, glycerol or glutamate as carbon source.

In connection with the third special embodiment of the cell according to the invention, it is especially preferred when this cell features an activity of at least one of the following enzymes E₁₀ to E₁₂, E₅₆, E₇₂ and E₇₃ which is increased in comparison with its wild type (see FIG. 15):

-   -   of an enzyme E₇₂, which catalyzes the conversion of beta-alanine         to beta-alanyl-coenzyme A,     -   of an enzyme E₇₃, which catalyzes the conversion of         beta-alanyl-coenzyme A into acrylyl-coenzyme A,     -   of an enzyme E₅₆, which catalyzes the conversion of         acrylyl-coenzyme A into methylmalonyl-coenzyme A,     -   of an enzyme E₁₀, which catalyzes the conversion of         methylmalonyl-coenzyme A into methyl malonate;     -   of an enzyme E₁₁, which catalyzes the conversion of methyl         malonate into methylmalonate-semialdehyde;     -   of an enzyme E₁₂, which catalyzes the conversion of         methylmalonate-semialdehyde into 3-hydroxyisobutyric acid.

In this context, cells which are especially preferred according to the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₅₆E₁₀, E₅₆E₁₁, E₅₆E₁₂, E₅₆E₁₀E₁₁ and E₇₂E₇₃E₅₆E₁₀E₁₁E₁₂. In connection with the fourth special embodiment, too, of the cell according to the invention it may be advantageous to overexpress an enzyme which is capable of catalyzing at least two of the above-described reaction steps. Here too, it is possible for example to employ an enzyme which features both the activity of the enzyme E₁₀ and the activity of the enzyme E₁₁, such as, for example, the malonyl-coenzyme A reductase from Sulfolobus tokodaii, which is encoded by the DNA sequence with the SEQ ID No 03 and which features the amino acid sequence as shown in SEQ ID No 04. Furthermore, it is, in principle, also possible in the context of the fourth special embodiment of the cell according to the invention to employ a cell which is already capable of forming especially large amounts of acrylyl-coenzyme A.

In this context, it is especially preferred that the enzyme

-   E₇₂ is a coenzyme A transferase (EC 2.8.3.1) or a coenzyme A     synthetase, preferably a coenzyme A transferase, -   E₇₃ is a beta-alanyl-coenzyme A ammonia-lyase (EC 4.3.1.6), -   E₅₆ is a crotonyl-coenzyme A decarboxylase -   E₁₀ is a methylmalonyl-coenzyme A hydrolase (EC 3.1.2.17), -   E₁₁ is an aldehyde dehydrogenase (EC 1.2.1.3) or an aldehyde oxidase     (EC 1.2.3.1) and -   E₁₂ is a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or a     3-hydroxyacyl-coenzyme A dehydrogenase (EC 1.1.1.35).

Preferred enzymes E₇₂ with a CoA transferase activity are those from Megasphaera elsdenii, Clostridium propionicum, Clostridium kluyveri and also from Escherichia coli. Examples which may be mentioned at this point of a DNA sequence coding for a CoA transferase is the sequence from Megasphaera elsdenii referred to as SEQ ID No: 24 in WO-A-03/062173. Enzymes which are furthermore preferred are those variants of the CoA transferase which are described in WO-A-03/062173.

Suitable enzymes E₇₃ with a beta-alanyl-coenzyme A ammonia-lyase activity are, for example, those from Clostridium propionicum. DNA sequences which code for such an enzyme can be obtained for example from Clostridium propionicum as described in Example 10 in WO-A-03/062173. The DNA sequence which codes for the beta-alanyl-coenzyme A ammonia-lyase from Clostridium propionicum is specified in WO-A-03/062173 as SEQ ID No: 22.

An enzyme E₅₆ which is preferably employed is, again, the crotonyl-coenzyme A decarboxylase from Rhodobacter sphaeroides, which is encoded by the DNA sequence with the SEQ ID No 05 and which features the amino acid sequence as shown in SEQ ID No 06. This enzyme is not only capable of converting crotonyl-coenzyme A into ethylmalonyl-coenzyme A, but also of converting acrylyl-coenzyme A into methylmalonyl-coenzyme A.

Suitable genes for the enzymes E₁₀ to E₁₂ have already been mentioned in connection with the first variant of the cell according to the invention, where it is also preferred in connection with the second variant, the above-described gene from Sulfolobus tokodaii is especially preferred as gene for the enzyme E₁₁.

According to an especially preferred variant of the third special embodiment of the cell according to the invention, this cell features at least one activity of the enzyme E₁₀ and E₅₆ or of the enzymes E₁₀, E₁₁ and E₅₆ which is increased in comparison with its wild type, where the E₁₀ or the enzymes E₁₀ and E₁₁ is encoded by a DNA sequence as shown in SEQ ID No 03 and the enzyme E₅₆ is encoded by a DNA sequence as shown in SEQ ID No 05. In this context, it is preferred when the increased activity of these two enzymes is achieved by overexpressing, in the cell, the polypeptides with SEQ ID No 04 and SEQ ID No 06 or else that amino acid sequences with at least 50%, preferably at least 55%, more preferably at least 60%, more preferably at least 65% and most preferably at least 70% identity with the amino acid sequence as shown in SEQ ID No 04 and SEQ ID No 06, respectively. In this context, these two DNA sequences may be integrated into the genome of the cell or else be present on a vector inside the cell.

In connection with the above-described third special embodiment of the cell according to the invention, it may furthermore be advantageous when the cell features not only an increase in the activity of the enzyme E₅₆ and/or of the activity of the enzyme E₁₀ or of the enzymes E₁₀ and E₁₁, but at least one, preferably both, of the following properties:

-   -   an activity of an enzyme E₁₁, which catalyzes the conversion of         pyruvate into oxaloacetate or of an enzyme E₇₄, which catalyzes         the conversion of phosphoenolpyruvate into oxaloacetate, but         preferably of an enzyme E₁₁, which catalyzes the conversion of         pyruvate into oxaloacetate, which is increased in comparison         with its wild type, and     -   an increased activity of an enzyme E₇₅, which catalyzes the         conversion of aspartate into beta-alanine.

The enzyme E₁₁ preferably takes the form of a carboxylase, especially preferably of a pyruvate carboxylase (EC number 6.4.1.1), which catalyzes the conversion of pyruvate into oxaloacetate. A pyruvate carboxylase which is especially preferred in this context is the mutant which is described in “A novel methodology employing Corynebacterium glutamicum genome information to generate a new L-lysine-producing mutant.” Ohnishi J et al., Applied Microbiology and Biotechnology, Vol. 58 (2), pages 217-223 (2002). In this mutation, the amino acid proline at position 458 has been substituted by serine. The disclosure of this publication with regard to the possibilities of preparing pyruvate carboxylate mutants is hereby incorporated by referent and forms part of the disclosure of the present invention.

The enzyme E₇₅ preferably takes the form of a decarboxylase, especially preferably of a glutamate decarboxylate or of an aspartate decarboxylase, with a 1-aspartate 1-decarboxylase (EC number 4.1.1.11) which is encoded by the panD gene being most preferred. Aspartate decarboxylase catalyzes the conversion of aspartate into beta-alanine. Genes for aspartate decarboxylase (panD genes) from, inter alia, Escherichia coli (FEMS Microbiology Letters, 143, pages 247-252 (1996)), “Photorhabdus luminescens subsp. Laumondii, Mycobacterium bovis subsp. Bovis”) and from a large number of other microorganisms have already been cloned and sequenced. DE-A-198 55 313 describes in particular the nucleotide sequence of the panD gene from Corynebacterium glutamicum. In principle, it is possible to use panD genes of any feasible origin, no matter whether from bacteria, yeasts or fungi. Furthermore, it is possible to employ all alleles of the panD gene, in particular also those which are the result of the degeneracy of the genetic code or of function-neutral sense mutations. An aspartate decarboxylase which is especially preferred according to the invention, besides the aspartate decarboxylase from Corynebacterium glutamicum, is the Escherichia coli mutant DV9 (Vallari and Rock, Journal of Bacteriology, 164, pages 136-142 (1985)). The disclosure of this publication with regard to the abovementioned mutant is hereby incorporated by reference and forms part of the disclosure of the present invention. The preparation of recombinant cells in which both the activity of the pyruvate carboxylase and the activity of the aspartate decarboxylase is increased is described in DE-A-10 2005 048 818.

According to a second variant of the cell according to the invention, the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor.

In the event that, in the cell according to the invention, the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor, as specified in the second variant, it is preferred according to a first special embodiment that the formation of 3-hydroxyisobutyric acid or of the polyhydroxyalkanoate based on 3-hydroxyisobutyric acid takes place via isobutyryl-coenzyme A as intermediate, where the cell is capable of preferentially utilizing carbohydrates, glycerol or L-valine as carbon source.

In the event that carbohydrates or glycerol act as the carbon source, it is preferred, according to a first alternative of this first special embodiment of the second variant of the cell according to the invention that this cell features an activity of at least one of the following enzymes E₇₆ to E₇₉, E₆₀, E₆₁ and E₈ which is increased in comparison with its wild type (see FIG. 16):

-   -   of an enzyme E₇₆, which catalyzes the conversion of pyruvate         into 2-acetolactate;     -   of an enzyme E₇₇, which catalyzes the conversion of         2-acetolactate into 2,3-dihydroxyisovalerate;     -   of an enzyme E₇₈, which catalyzes the conversion of         2,3-dihydroxyisovalerate into 2-oxoisovalerate;     -   of an enzyme E₇₉, which catalyzes the conversion of         2-oxoisovalerate into isobutyryl-coenzyme A;     -   of an enzyme E₆₀, which catalyzes the conversion of         isobutyryl-coenzyme A into methacrylyl-coenzyme A;     -   of an enzyme E₆₁, which catalyzes the conversion of         methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;     -   of an enzyme E₈, which catalyzes the conversion of         3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₈, E₆₀, E₆₁, E₇₆, E₇₇, E₇₈, E₇₉ and E₈E₆₀E₆₁E₇₆E₇₇E₇₈E₇₉.

In this context, it is especially preferred that the enzyme

-   E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), -   E₇₆ is an acetolactate synthase (EC 2.2.1.6), -   E₇₇ is a dihydroxyisovalerate dehydrogenase (EC 1.1.1.86), -   E₇₈ is a 2,3-dihydroxyisovalerate dehydratase (EC 4.2.1.9), -   E₇₉ is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4), -   E₆₀ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a     butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a     2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12), and -   E₆₁ is an enoyl-coenzyme A hydratase (EC 4.2.1.17).

Preferred enzymes E₈, E₆₀ and E₆₁ are those which have already been described above.

The enzyme E₇₆ is preferably encoded by genes selected from the group consisting of ilvb1, t8p19.70, ilv1, ilv2, ilv6, aa1021wp, ae1305cp, ilvI, ilvH, ilvN, ilvB, ilvM, ilvG, ilvN, budB, ilvN-1, ilvN-2, atrC, ilvX, iolD, budB, alsS, ilvK, ilvB1, ilvB2, ilvB3, ilvN1, ilvN2, cgl1271, cgl1272, iolD and scc57A.40c.

The enzyme E₇₇ is preferably encoded by genes selected from the group consisting of f14p22.200, ilv5, ac1198Wp, ilvC, ilvY, ilvC-1, ilvC-2, ilvC-3 and cgl1273, where the ilvC gene is most preferred.

The enzyme E₇₈ is preferably encoded by genes selected from the group consisting of f14o13.18, ilv3, acl117wp, ilvD, cgl1268, ilvD1 and ilvD2, where ilvD is most preferred.

In the event that L-valine acts as carbon source, it is preferred according to a second modification of the first special embodiment of the second alternative of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor and isobutyryl-coenzyme A as intermediate, that this cell features an activity of at least one of the following enzymes E₇₉, E₈₀, E₆₀, E₆₁ and E₈ which is increased in comparison with its wild type (see FIG. 17):

-   -   of an enzyme E₈₀, which catalyzes the conversion of L-valine         into 2-oxoisovalerate;     -   of an enzyme E₇₉, which catalyzes the conversion of         2-oxoisovalerate into isobutyryl-coenzyme A;     -   of an enzyme E₆₀, which catalyzes the conversion of         isobutyryl-coenzyme A into methacrylyl-coenzyme A;     -   of an enzyme E₆₁, which catalyzes the conversion of         methacrylyl-coenzyme A into 3-hydroxyisobutyryl-coenzyme A;     -   of an enzyme E₈, which catalyzes the conversion of         3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₈, E₆₀, E₆₁, E₇₉, E₈₀ and E₈E₆₀E₆₁E₇₉E₈₀.

In this context, it is especially preferred that the enzyme

-   E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), -   E₆₀ is an acyl-coenzyme A dehydrogenase (EC 1.3.99.3), a     butyryl-coenzyme A dehydrogenase (EC 1.3.99.2) or a     2-methylacyl-coenzyme A dehydrogenase (EC 1.3.99.12), -   E₆₁ is an enoyl-coenzyme A hydratase (EC 4.2.1.17), -   E₇₉ is a 2-oxoisovalerate dehydrogenase (EC 1.2.1.25 or EC 1.2.4.4),     and -   E₈₀ is an amino acid transferase (EC 2.6.1.42),

Preferred enzymes E₈, E₆₀, E₆₁ and E₇₉ are those which have already been described above.

The enzyme E₈₀ is preferably encoded by genes selected from the group consisting of bcat1, bcat2, t27I1.8, t27i1.9, f2j10.5, f2j10.4, t12h1.16, mmb12.20, t9c5.3, mpa24.13, bat1, bat2, ad1384wp, eca39, bcaA, ilvE, ilvE1, ilvE2, ilvE3, ywaA, ybgE, bcaT and cgl2204, where ilvE is especially preferred.

The nucleotide sequences of suitable genes the enzyme E₈₀ can, again, be found in the KEGG database, the NCBI database or the EMBL database.

In connection with this second alternative of the first special embodiment of the second variant of the cell according to the invention, it may furthermore be advantageous to reduce the activity of an enzyme E₄ which catalyzes the conversion of methylmalonate-semialdehyde into 3-hydroxyisobutyric acid, where this enzyme E₄ preferably takes the form of a 3-hydroxyisobutyrate dehydrogenase (EC 1.1.1.31) or of a 3-hydroxy-acyl-coenzyme A dehydrogenase (EC 1.1.1.35).

According to the second modification of the first special embodiment of the second variant of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor and isobutyryl-coenzyme A as intermediate and starting from L-valine as carbon source, it may furthermore be preferred to employ those cells which are already capable of forming large amounts of L-valine. In this context, suitable cells are in particular those which have been described by Blombach et al. in Applied Environmental Microbiology, Vol. 73 (7) (2007), pages 2079-2084.

In the event that C₁-compounds such as, for example, methane or methanol act as carbon source, it is preferred in a second special embodiment of the second variant of the cell according to the invention, where the formation of 3-hydroxyisobutyric acid or of polyhydroxyalkanoates based on 3-hydroxyisobutyric acid takes place via 3-hydroxyisobutyryl-coenzyme A as precursor, that the formation takes place via 3-hydroxyisobutyryl-coenzyme A as intermediate. In this context, it is preferred that the cell features an activity of at least one of the following enzymes E₈, E₅₃, E₅₄ and E₈₁ which is increased in comparison with its wild type:

-   -   of an enzyme E₅₃, which catalyzes the conversion of         acetyl-coenzyme A into acetoacetyl-coenzyme A;     -   of an enzyme E₅₄, which catalyzes the conversion of         acetoacetyl-coenzyme A into 3-hydroxybutyryl-coenzyme A;     -   of an enzyme E₈₁, which catalyzes the conversion of         3-hydroxybutyryl-coenzyme A into 3-hydroxy-isobutyryl-coenzyme         A;     -   of an enzyme E₈, which catalyzes the conversion of         3-hydroxyisobutyryl-coenzyme A into 3-hydroxyisobutyrate.

In this context, genetically modified cells which are especially preferred in accordance with the invention are those in which the activity of the following enzymes or enzyme combinations is increased: E₈, E₅₃, E₅₄, E₈₁ and E₈E₅₃E₅₄E₈₁.

In this context, it is especially preferred that the enzyme

-   E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase (EC 3.1.2.4), -   E₅₃ is a β-ketothiolase (EC 2.3.1.9), -   E₅₄ is an acetoacetyl-coenzyme A reductase (an EC 1.1.1.36), and -   E₈₁ is an isobutyryl-coenzyme mutase (EC 5.4.99.13).

Preferred enzymes E₈, E₅₃ and E₅₄ are those which have already been described hereinabove. A preferred enzyme E₈₁ is the isobutyryl-coenzyme mutase from β-proteo-bacterium strain L108 which is described in Applied And Environmental Microbiology, Vol. 72 (6), 2006, pages 4128-4135.

According to a special embodiment of the cell according to the invention, it is furthermore preferred that this cell features an expression of the glb0 gene which is increased in comparison with its wild type. Furthermore, it may under certain circumstances be preferred that the cell according to the invention features an activity of the citrate transport protein which is encoded by the dctA gene or the citP gene, which activity is reduced in comparison with its wild type.

A contribution to the solution of the problems mentioned at the outset is furthermore provided by a method of preparing a genetically modified cell which is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid via methylmalonate-semialdehyde or isobutyryl-coenzyme A, as precursors, comprising the method step of increasing, in the cell, the activity of at least one of the above-described enzymes, preferably of one or more of the enzymes

-   -   E₁ to E₄,     -   E₁, E₄, E₅, E₆ and E₇,     -   E₁, E₄, E₅ and E₇,     -   E₄, E₅ and E₄₇ to E₅₂,     -   E₂ to E₄, E₆, E₇ and E₄₇ to E₅₂,     -   E₂ to E₄, E₇ and E₄₇ to E₅₂,     -   E₈ and E₅₃ to E₆₁,     -   E₈, E₆₀, E₆₁ and E₇₆ to E₇₉,     -   E₈, E₆₀, E₆₁, E₇₉ and E₈₀, or     -   E₈, E₅₃, E₅₄ and E₈₂         in the cell, where increasing the enzymatic activity is         preferably carried out by the methods described at the outset.

Another contribution to the solution of the problems mentioned at the outset is provided by the cells obtainable by the above-described method.

Another contribution to the solution of the problems mentioned at the outset is provided by a method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, comprising the method step of bringing a cell according to the invention into contact with a nutrient medium comprising, as carbon source, carbohydrates, glycerol, carbon dioxide, methane, methanol, L-valine or L-glutamate under conditions under which 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid are formed from the carbon source, and, if appropriate, isolation of the 3-hydroxyisobutyric acid from the nutrient medium.

The genetically modified cells according to the invention can be into contact with the nutrient medium, and thus cultured, either continuously or batchwise in the batch method or in the fed-batch method or in the repeated-fed-batch method in order to produce 3-hydroxyisobutyrate or polyhydroxyalkanoates based on 3-hydroxyisobutyrate. A semicontinuous method as described in GB-A-1009370 is also feasible. An overview over known culture methods are described in the textbook by Chmiel (“Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik” [Bioprocess technology 1. introduction to bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (“Bioreaktoren und periphere Einrichtungen”, [Bioreactors and peripheral equipment] Vieweg Verlag, Braunschweig/Wiesbaden, 1994).

The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981).

Carbon sources which may be used are carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats such as, for example, soy oil, sunflower oil, peanut oil and coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and linolic acid, alcohols such as, for example, glycerol and methanol, hydrocarbons such as methane, amino acids such as L-glutamate or L-valine, or organic acids such as, for example, acetic acid. These substances may be used singularly or as a mixture. It is especially preferred to employ carbohydrates, in particular monosaccharides, oligosaccharides or polysaccharides, as described in U.S. Pat. No. 601,494 and U.S. Pat. No. 6,136,576, or C₅-sugars, or glycerol.

Nitrogen sources which can be used are organic nitrogen-comprising compounds such as peptones, yeast extract, meat extract, malt extract, cornsteep liquor, soya mill and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources can be used singularly or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-comprising salts can be used as sources of phosphorus. The culture medium must furthermore comprise salts of metals such as, for example, magnesium sulfate or iron sulfate, which are required for growth. Finally, essential growth factors such as amino acids and vitamins may be employed in addition to the abovementioned substances. Moreover, suitable precursors may be added to the culture medium. The abovementioned input materials may be added to the culture in the form of a single batch or else fed in a suitable manner during culturing.

The pH for the culture can be controlled by employing, in an appropriate manner, basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia, or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters. To maintain the stability of plasmids, it is possible to add to the medium suitable substances which have a selective effect, such as, for example, antibiotics. Aerobic conditions are maintained by introducing, into the culture, oxygen or oxygen-containing gas mixtures such as, for example, ambient air. The culture temperature is normally 20° C. to 45° C. and preferably 25° C. to 40° C. It may be preferred to employ, as cells, those cells which are described in U.S. Pat. No. 6,803,218, in particular when using cells which are capable of converting glycerol as the substrate. In this case, the cells can be cultured at temperatures in the range of from 40 to 100° C.

The isolation of 3-hydroxyisobutyric acid from the nutrient solution is preferably carried out continuously, it being furthermore preferred in this context also to produce 3-hydroxyisobutyric acid by fermentation in a continuous manner, so that the entire process from the production of 3-hydroxyisobutyric acid up to its isolation from the fermentation liquor can be carried out continuously. For the continuous isolation of the production of 3-hydroxyisobutyric acid from the fermentation liquor, the former is continuously passed over a device for removing the microorganisms employed during fermentation, preferably through a filter with an exclusion level in the range of from 20 to 200 kDa, where a solid/liquid separation takes place. It is also feasible to employ a centrifuge, a suitable sedimentation device or a combination of these devices, it being especially preferred to first separate at least part of the microorganisms by sedimentation and subsequently to feed the fermentation liquor, which has been freed from part of the microorganisms, to ultrafiltration or to a centrifugation device.

After the microorganisms have been removed, the fermentation product, which is enriched with regard to its 3-hydroxyisobutyric acid fraction, is fed to a separation system, preferably a multistep separation system. This separation system provides a plurality of separation steps which are connected in series, from which steps in each case return lines lead away and back to the fermentation tank. Furthermore, exit pipes lead out of the respective separation steps. The individual separation steps may operate by the electrodialysis, the reverse osmosis, the ultrafiltration or the nanofiltration principle. As a rule, these are membrane separation devices in the individual separation steps. The selection of the individual separation steps is a function of the nature and the extent of the fermentation by-products and substrate residues.

Besides the 3-hydroxyisobutyric acid being separated off by means of electrodialysis, reverse osmosis, ultrafiltration or nanofiltration, in the course of which an aqueous 3-hydroxyisobutyric acid solution is obtained as the end product, the 3-hydroxyisobutyric acid can also be separated off by extractive methods from the fermentation solution which has been freed from microorganisms, in which case, finally, the pure 3-hydroxyisobutyric acid can be obtained. To separate the 3-hydroxyisobutryic acid by extraction, it is possible to add, to the fermentation solution, for example ammonium compounds or amines in order to form an ammonium salt of 3-hydroxyisobutyric acid. This ammonium salt can then be separated from the fermentation solution by adding an organic extractant and subsequently heating the resulting mixture, whereby the ammonium salt is concentrated in the organic phase. Then, the 3-hydroxyisobutyric acid can be isolated from this phase for example by further extraction steps, giving the pure 3-hydroxyisobutyric acid. More details regarding the separation method can be found in WO-A-02/090312, whose disclosure regarding the separation of hydroxycarboxylic acids from fermentation solutions is hereby incorporated by reference and forms part of the disclosure of the present application.

Depending on the way in which the 3-hydroxyisobutyric acid is separated from the fermentation solution, either an aqueous solution of 3-hydroxyisobutyric acid comprising 2 to 90% by weight, preferably 7.5 to 50% by weight and especially preferably 10 to 25% by weight of 3-hydroxyisobutyric acid, or else pure 3-hydroxyisobutyric acid is obtained.

Furthermore, the 3-hydroxyisobutyric acid prepared by the method according to the invention can also be neutralized, either before, during or after the purification, for which purpose bases such as, for example, calcium hydroxide or sodium hydroxide can be employed.

A contribution to solving the problems mentioned at the outset is provided in particular also by a method of preparing methacrylic acid or methacrylic esters, comprising the method steps

-   IA) preparation of 3-hydroxyisobutyric acid by the method described     above and, if appropriate, isolation and/or neutralization of the     3-hydroxyisobutyric acid of the 3-hydroxyisobutyric acid, -   IB) dehydration of the 3-hydroxyisobutyric acid with formation of     methacrylic acid and, if appropriate, esterification of the     methacrylate or of the methacrylic acid.

According to method step IB), the 3-hydroxyisobutyric acid is dehydrated with formation of methacrylic acid, for which purpose it is possible either to employ the pure 3-hydroxyisobutyric acid isolated from the fermentation solution or else the aqueous solution of 3-hydroxyisobutryic acid, which has been isolated when working up the fermentation solution, it also being possible to concentrate the aqueous solution of 3-hydroxyisobutyric acid, if appropriate, before the dehydration step, for example by means of distillation, if appropriate in the presence of a suitable entrainer.

The dehydration reaction can, in principle, be carried out in liquid phase or in the gas phase. Furthermore, it is preferred in accordance with the invention that the dehydration reaction is carried out in the presence of a catalyst, with the nature of the catalyst employed depending on whether a gas-phase or a liquid-phase reaction is carried out.

Suitable dehydration catalysts are both acidic catalysts and alkaline catalysts. Acidic catalysts are preferred, in particular because they show less tendency to form oligomers. The dehydration catalyst may be employed both as a homogeneous and as a heterogeneous catalyst. If the dehydration catalyst is present in the form of a heterogeneous catalyst, it is preferred that the dehydration catalyst is in contact with a support x. Suitable supports x are all solids believed by the skilled worker to be suitable. In the present context, it is preferred that the solids have suitable pore volumes which are suitable for good binding and absorption of the dehydration catalyst. Furthermore, total pore volumes as specified by DIN 66133 in a range of from 0.01 to 3 ml/g are preferred, and total pore volumes in the range of from 0.1 to 1.5 ml/g are especially preferred. Moreover, it is preferred that the solids which are suitable as support x have a surface area in the range of from 0.001 to 1000 m²/g, preferably in the range of from 0.005 to 450 m²/g and furthermore preferred in the range of from 0.01 to 300 m²/g as determined by BET test as specified in DIN 66131. A support which may be employed for the dehydration catalyst can firstly be bulk material with a mean particle diameter in the range of from 0.1 to 40 mm, preferably in the range of from 1 to 10 mm, and furthermore preferably in the range from 1.5 to 5 mm. The wall of the dehydration reactor may furthermore act as support. Furthermore, the support may be acidic or alkaline per se, or else an acidic or alkaline dehydration catalyst may be applied to an inert support. Application techniques which may be mentioned in particular are immersion or impregnation or else incorporation into a support matrix.

Suitable supports x, which may also feature dehydration catalyst properties, are, in particular, natural or synthetic silicates such as, in particular, mordenite, montmorillonite, acidic zeolites; supports which are coated with monobasic, dibasic or polybasic inorganic acids, in particular phosphoric acid, or with acidic salts of inorganic acids, such as substances of the oxide or silicate type, for example Al₂O₃, TiO₂; oxides and mixed oxides such as, for example, γ-Al₂O₃ and ZnO—Al₂O₃ mixed oxides of the heteropolyacids.

In accordance with an embodiment according to the invention, the support x consists at least in part of a compound of the oxide type. Such compounds of the oxide type should feature at least one of the elements selected from among Si, Ti, Zr, Al, P or a combination of at least two of these. Such supports may also act as dehydration catalyst themselves, owing to their acidic or alkaline properties. A preferred class of compounds, both as support by way of x and by way of dehydration catalyst comprise silicon/aluminum/phosphorus oxides. Preferred alkaline substances which act both as dehydration catalyst and also as support x comprise alkali, alkaline earth, lanthanum, lanthoids or a combination of at least two of these in the form of their oxides. Such acidic or alkaline dehydration catalysts are commercially available both from Degussa AG and from Südchemie AG. A further class are ion exchangers. Again, these may be present both in alkaline and in acidic form.

Suitable homogeneous dehydration catalysts are, in particular, inorganic acids, preferably phosphorus-containing acids and furthermore preferably phosphoric acid. These inorganic acids can be immobilized on the support x by immersion or impregnation.

The use of heterogeneous catalysts has proved particularly advantageous in particular in the case of gas phase dehydration. In the case of liquid-phase dehydration, however, both homogeneous and heterogeneous dehydration catalysts are employed.

Furthermore, it is preferred that the method according to the invention involves the use of a dehydration catalyst with an H₀ value in the range of from +1 to −10, preferably in the range of from +2 to −8.2 and furthermore preferably, in the case of liquid-phase dehydration, in the range of from +2 to −3 and in gas-phase dehydration in the range of from −3 to −8.2. The H₀ value corresponds to the acid function as defined by Hämmert and can be determined by what is known as amine titration and the use of indicators, or by the absorption of a gaseous base (see “Studies in Surface Science and Catalytics”, vol. 51, 1989: “New solid Acids and Bases, their catalytic Properties”, K. Tannabe et al).

According to a special embodiment of the method according to the invention, the acidic solid catalyst employed is a porous support structure which has been brought into contact with an inorganic acid, preferably with phosphoric acid or with superacids such as, for example, sulfated or phosphated zirconium oxide and which is based preferably on at least 90% by weight, furthermore preferably at least 95% by weight and most preferably at least 99% by weight of a silicon oxide, preferably an SiO₂. The bringing into contact of the porous support structure with the inorganic acid is preferably carried out by impregnating the support structure with the acid, with the latter preferably being brought into contact with the former in an amount in a range of from 10 to 70% by weight, especially preferably in the range of from 20 to 60% by weight and more preferably in a range of from 30 to 50% by weight, based on the weight of the support structure, followed by drying. After drying, the support structure is heated in order to fix the inorganic acid, preferably at a temperature in a range of from 300 to 600° C., more preferably in a range of from 400 to 500° C.

According to a special embodiment of the method according to the invention, the dehydration reaction is carried out in the gas phase. Here, it is possible to employ conventional apparatuses as are known to the skilled worker in the field of gas phase reaction, for example tubular reactors. It is especially preferred to employ shell-and-tube heat exchangers and reactors which comprise thermoplates as heat exchangers.

According to an embodiment of the gas-phase dehydration reaction, pure 3-hydroxyisobutyric acid is introduced into a reactor comprising one of the abovementioned fixed-bed catalysts. According to another embodiment, the 3-hydroxyisobutyric acid is introduced into the reactor in the form of an aqueous solution comprising 2 to 80% by weight, especially preferably 5 to 50% by weight and more preferably 10 to 25% by weight of 3-hydroxyisobutyric acid, in each case based on the total weight of the aqueous solution. The pressure and temperature conditions inside the reactor are chosen such that the 3-hydroxyisobutyric acid, or the aqueous solution, is present in gaseous form when entering the reactor. The dehydration in the gas phase is preferably carried out in the temperature range of between 200 and 400° C., especially preferably between 250 and 350° C. The pressure inside the reactor during the gas-phase dehydration reaction is preferably in a range of from 0.1 to 50 bar, especially preferably in a range of from 0.2 to 10 bar and most preferably in a range of from 0.5 to 5 bar.

The amount of 3-hydroxyisobutyric acid introduced into the reactor in the gas-phase dehydration reaction is preferably in a range of from 10 to 100% by volume, especially preferably in a range of from 20 to 100% by volume and most preferably in a range of from 30 to 100% by volume.

According to another special embodiment of the method according to the invention, the dehydration reaction is performed in the liquid phase. The liquid-phase dehydration reaction can also be carried out in all apparatuses which are known to the skilled worker and in which a fluid can be heated to a desired reaction temperature, during which process a pressure can be applied to the apparatus which is sufficient for maintaining the reaction components in the liquid state under the desired temperature conditions.

According to a special embodiment of the method according to the invention, the liquid-phase dehydration method comprises a first method step, in which pure 3-hydroxyisobutyric acid or an aqueous solution comprising 5 to 100% by weight, especially preferably 20 to 100% by weight and most preferably 50 to 100% by weight of 3-hydroxyisobutyric acid, based on the total weight of the aqueous solution, is introduced into a reactor. The pressure and temperature conditions inside the reactor are chosen such that the 3-hydroxyisobutyric acid, or the aqueous solution, is present in liquid form when entering the reactor. According to a special embodiment of the method according to the invention in which the dehydration reaction is carried out in the liquid phase, the 3-hydroxyisobutyric acid, or the aqueous solution, is passed in such a way over a fixed catalyst bed inside the dehydration reactor that the liquid phase trickles over the surface of the catalyst particles. Such a procedure may be carried out for example in a trickle-bed reactor.

The dehydration in the liquid phase is preferably carried out in a temperature range of between 200 and 350° C., especially preferably between 250 and 300° C. The pressure inside the reactor in the case of liquid-phase dehydration is preferably in a range of from 1 to 50 bar, especially preferably in a range of from 2 to 25 bar and most preferably in a range of from 3 to 10 bar.

The catalysis of the dehydration reaction may be homogeneous or heterogeneous, both in the case of gas-phase dehydration and in the case of liquid-phase dehydration.

In the case of homogeneous catalysis, the catalyst, which in this case preferably takes the form of an inorganic acid such as, for example, phosphoric acid or sulfuric acid, is first brought into contact with the pure 3-hydroxyisobutyric acid or with the aqueous solution comprising the 3-hydroxyisobutyric acid. Thereafter, the resulting composition is introduced into the reactor and converted into methacrylic acid under the desired pressure and temperature conditions. It is also feasible to introduce the inorganic acid independently of the 3-hydroxyisobutyric acid or the aqueous solution into the reactor. In this case, the reactor features at least two feed lines, one for the 3-hydroxyisobutyric acid, or the aqueous solution comprising 3-hydroxyisobutyric acid, and one for the catalyst. If the dehydration reaction is carried out in liquid phase in a trickle-bed reactor, it is preferred to introduce the catalyst together with the 3-hydroxyisobutyric acid, or the aqueous solution comprising the 3-hydroxyisobutyric acid, at the top of the reactor.

In the case of heterogeneous catalysis, the catalyst is in the form of a solid substrate located in the reaction space, for example in the form of a fixed bed, in the form of catalyst-coated plates, preferably thermoplates, which are arranged inside the reactor, or else in the form of catalyst-coated reactor walls. Reactors which are possible are described for example in DE-A-198 48 208, DE-A-100 19 381 and EP-A-I 234 612. In the case of heterogeneous catalysis, preferred catalysts are support structures which have been brought into contact with inorganic acids, preferably impregnated porous support structures. The 3-hydroxyisobutyric acid, or the aqueous solution comprising the 3-hydroxyisobutyric acid, is then brought into contact with the surface of the solid catalyst material in the form of a vapor, or in liquid form.

According to an especially preferred embodiment of the method according to the invention, the dehydration of the 3-hydroxyisobutyric acid is carried out in liquid phase at a pressure in the range of from 200 to 500 mbar, at a temperature in a range of from 200 to 230° C. and in the presence of alkali metal ions as the catalyst.

The reaction mixture which is obtained after the dehydration reaction is either an aqueous methacrylic acid solution which does not contain any catalyst components (such a solution is obtained in the case of heterogeneously catalyzed dehydration) or else an aqueous methacrylic acid solution which comprises catalysts (such a solution is obtained in the case of homogeneously catalyzed dehydration). Furthermore, the aqueous methacrylic acid solution may be in liquid form (if the dehydration reaction has been effected in the liquid phase) or in gaseous form (if the dehydration reaction has been carried out in the gas phase).

If appropriate, the resulting methacrylic acid solution can, according to a special embodiment of the method according to the invention, be esterified without further processing. In such a case, the methacrylic acid solution is brought into contact with suitable alcohols such as, for example, methanol, ethanol, 1-propanol, 2-propanol or 1-butanol and suitable esterification catalysts known to the skilled worker such as, for example, concentrated acids, with heating, and the methacrylic acid is so converted into the corresponding esters. However, it may be advantageous additionally to purify the methacrylic acid before esterification, it being possible to employ, in principle, any purification method which is known to the skilled worker and which is conventionally employed for the purification of contaminated (meth)acrylic acid obtained by catalytic gas-phase oxidation of propylene.

If the dehydration reaction has been carried out in the gas phase, it is preferred that the methacrylic acid is first condensed, generating an aqueous methacrylic acid solution. Here, any condensation method known to the skilled worker may be employed in principle, for example a fractional condensation as described in WO-A-2004/035514, WO-A-03/014172 or EP-A-EP 1 163 201 or by total condensation as described in EP-A-0 695 736. It is also feasible to add additional solvents, in particular water, during the condensation process in order to absorb the methacrylic acid as completely as possible.

The aqueous methacrylic acid solution obtained after condensation, or else the aqueous methacrylic acid solution obtained in the event of liquid-phase dehydration, can then be freed from water and other contaminants in further purification steps. Here, it is possible first to remove the water by azeotrope distillation in the presence of an entrainer as described, for example, in DE-A-198 53 064. It is also feasible to employ high-boiling organic solvents for absorbing the methacrylic acid, as is disclosed for example in EP-A-0 974 574. In addition to these distillation methods, membranes for dewatering may also be employed, as proposed for example in DE-A-44 01 405. Employing crystallization methods for purifying the aqueous methacrylic acid solution, which has been generated in the case of liquid-phase dehydration or which has been obtained by condensation, is furthermore feasible.

The methacrylic acid obtained after dehydration can be purified even further in further method steps. Thus, high-boiling contaminants which are still present can be removed by further distillation steps. However, it is especially preferred to further purify the methacrylic acid obtained by dehydration using crystallization methods as described for example in DE-A-101 49 353.

The resulting purified methacrylic acid can then be esterified, if appropriate.

A contribution to solving the problems mentioned at the outset is furthermore provided by a method of preparing methacrylic acid or methacrylic esters, comprising the method steps

-   IIA) preparation of polyhydroxyalkanoates based on     3-hydroxyisobutyric acid by the method described above, -   IB) cleavage of the polyhydroxyalkanoates based on     3-hydroxyisobutyric acid with formation of 3-hydroxyisobutyric acid     and, if appropriate, neutralization of the 3-hydroxyisobutyric acid     and/or isolation of the 3-hydroxyisobutyric acid, -   IIC) dehydration of the 3-hydroxyisobutyric acid with formation of     methacrylic acid and, if appropriate, esterification of the     methacrylate or methacrylic acid.

A contribution to solving the problems mentioned at the outset is also provided by a method of preparing polymethacrylic acid or polymethacrylic esters, comprising the method steps

-   IIIA) preparation of methacrylic acid by the method described above, -   IIIB) free-radical polymerization of the methacrylic acid,     it being possible, if appropriate, to esterify at least in part the     carboxyl groups of the methacrylic acid before or after the     free-radical polymerization reaction.

A contribution to solving the problem mentioned at the outset is furthermore provided by an isolated DNA, which is selected from the following sequences:

-   a) a sequence as shown in SEQ ID No 03, -   b) an intron-free sequence which is derived from a sequence as     specified in a) and which codes for the same protein or peptide as     the sequence as shown in SEQ ID No 03, -   c) a sequence which codes for a protein or peptide which comprises     the amino acid sequence as shown in SEQ ID No 04, -   d) a sequence with at least 80%, especially preferably at least 90%,     more preferably at least 95% and most preferably 99% identity with a     sequence as specified in one of groups a) to c), especially     preferably as specified in group a), this sequence preferably coding     for a protein or peptide which is capable of converting both S- or     R-methylmalonyl-coenzyme A and malonyl-coenzyme A into the     corresponding semialdehydes ((S)- or (R)-methylmalonate semialdehyde     and malonate semialdehyde, respectively), -   e) a sequence which hybridizes, or, taking into consideration the     degeneration of the genetic code, would hybridize, with the counter     strain of a sequence as specified in any of groups a) to d),     especially preferably as specified in group a), this sequence     preferably coding for a protein or peptide which is capable of     converting both S- or R-methylmalonyl-coenzyme A and     malonyl-coenzyme A into the corresponding semialdehydes ((S)- or     (R)-methylmalonate semialdehyde and malonate semialdehyde,     respectively), -   f) a derivative of a sequence as specified in any of groups a) to     e), especially preferably as specified in group a), this derivative     preferably coding for a protein or peptide which is capable of     converting both S- or R-methylmalonyl-coenzyme A and     malonyl-coenzyme A into the corresponding semialdehydes ((S)- or     (R)-methylmalonate semialdehyde and malonate semialdehyde,     respectively), obtained by substitution, addition, inversion and/or     deletion of at least one base, preferably of at least 2 bases, more     preferably of at least 5 bases and most preferably at least 10     bases, but preferably of no more than 100 bases, especially     preferably of no more than 50 bases and most preferably of no more     than 25 bases, and -   g) a sequence which is complementary to a sequence as specified in     any of groups a) to f), especially preferably as specified in group     a).

Surprisingly, it has been found that a DNA which has been isolated from bacteria of the strain Sulfolobus tokodaii (at the Deutsche Sammlung von Mikroorganismen [German collection of microorganisms], deposit number DSM 16993) and which has a DNA sequence as shown in SEQ ID No 03 codes for a polypeptide (SEQ ID No 04) which is capable even at temperatures of up to 75° C. of converting both S- or R-methylmalonyl-coenzyme A and malonyl-coenzyme A into the corresponding semialdehydes ((S)- or (R)-methylmalonate semialdehyde and malonate semialdehyde, respectively). Since (S)- or (R)-methylmalonate semialdehyde and malonate semialdehyde are natural metabolites which are formed for example during the degradation of valine, of leucin or of isoleucin, during the propanoate metabolism or during the pyruvate metabolism, and because the formed semialdehydes are capable of being reduced further in the course of the abovementioned metabolic pathways to give the corresponding 3-hydroxyalkanoates, the isolated DNA according to the invention can be utilized for generating recombinant bacteria which are capable of directly forming large amounts of 3-hydroxyisobutyric acid (or 3-hydroxypropionic acid). If the cells are furthermore capable of polymerizing the formed 3-hydroxyalkanoates with formation of polyhydroxyalkanoates, this DNA would furthermore be suitable for generating recombinant bacteria capable of producing polyhydroxyalkanoates based on 3-hydroxyisobuytric acid (or on 3-hydroxypropionic acid).

The “nucleotide identity” in relation to SEQ ID No 03, which is defined in alternative d), is determined with the aid of known methods here. In general, specialist computer programs with algorithms taking into consideration specific requirements are used.

Preferred methods of determining the identity first generate the maximum agreement between the sequences to be compared. Computer programs for determining the identity comprise the GCG program package, including but not limited thereto

-   -   GAP (Deveroy, J. et al., Nucleic Acid Research 12 (1984), page         387, Genetics Computer Group University of Wisconsin, Medicine         (Wi)), and     -   BLASTP, BLASTN and FASTA (Altschul. S. et al., Journal of         Molecular Biology 215 (1990), pages 403-410). The BLAST program         may be obtained from the Center For Biotechnology Information         (NCBI) and from other sources (BLAST Manual, Altschul S. et al.,         NCBI NLM NIH Bethesda ND 22894; Altschul S. et al., above).

The Smith-Waterman algorithm, which is known, can also be used for determining the nucleotide identity.

Preferred parameters for the nucleotide alignment comprise the following:

-   -   Algorithmus Needleman and Wunsch, Journal of Molecular Biology         48 (1970), pages 443-453     -   alignment matrix

Matches = +10 Mismatches = 0 Gap penalty = 50 Gap length penalty = 3

The GAP program is also suitable for use with the above parameters. The above parameters are the default parameters in the nucleotide sequence alignment.

An identity of 80% according to the above algorithm means 80% identity in the context of the present invention. The same applies to greater identities.

The feature “sequence which hybridizes, or, taking into consideration the degeneracy of the genetic code, would hybridize, with the counter strain of a sequence as specified in one of groups a) to d), especially preferably as specified in group a),” according to alternative e) indicates a sequence which hybridizes, or would hybridize taking into consideration the degeneracy of the genetic code, with the counter strand of a sequence as specified in one of groups a) to d), especially preferably as specified in group a), under preferably stringent conditions. For example, the hybridization reactions can be carried out at 68° C. in 2×SSC, or as described in the protocol of the dioxygenin labeling kit from Boehringer (Mannheim). Examples of preferred hybridization conditions are incubation overnight at 65° C. in 7% SDS, 1% BSA, 1 mM EDTA, 250 mM sodium phosphate buffer (pH 7.2), followed by washing at 65° C. with 2×SSC; 0.1% SDS.

The derivatives, of the isolated DNA according to the invention, which can be obtained according to alternative f) by substitution, addition, inversion and/or deletion of one or more bases of a sequence as specified in any of groups a) to e) include in particular those sequences which, in the protein which they encode, lead to conservative amino acid substitutions such as, for example, the substitution of glycine for alanine or of aspartate for glutamic acid. Such function-neutral mutations are referred to as sense mutations and do not lead to any principle modification of the activity of the polypeptide. It is furthermore known that modifications at the N and/or C terminus of a polypeptide do not have a considerable adverse effect on its function; indeed, they are even capable of stabilizing it, so that, as a consequence, the present invention also comprises DNA sequences where bases are added at the 3′ terminus or at the 5′ terminus of the sequence with the SEQ ID No 03. The skilled worker will find information on this subject in Ben Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O'Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6: 1321-1325 (1988)) inter alia, and in known textbooks of Genetics and Molecular Biology.

To isolate the DNA according to the invention, an NADPH-dependant malonyl-coenzyme A reductase was first isolated from a cell extract of Metallosphaera sedula and purified. The first 20 amino acids of the N terminus of the polypeptide of the resulting purified enzyme were sequenced. The gene for the malonyl-coenzyme A reductase was subsequently determined in the genome of Sulfolobus tokodaii, which has already been fully sequenced (Kawarabayasi et al., “Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.”, DNA Research 8:123-40), by identifying the derived protein sequence which is identical with the first 20 amino acids of the polypeptide isolated from Metallosphaera sedula. The DNA sequence according to the invention was then amplified by means of PCR, using suitable primers (see example 2).

A contribution to solving the problems mentioned at the outset is furthermore contributed by a vector, preferably an expression vector, comprising a DNA with a sequence as specified in one of groups a) to f) as defined above. Suitable vectors are all vectors which are known to the skilled worker and which are traditionally employed for introducing DNA into a host cell. Preferred vectors are selected from the group consisting of plasmids, such as, for example, the E. coli plasmids pTrc99A, pBR345 and pBR322, viruses such as, for example, bacteriophages, adenoviruses, vaccinia viruses, baculoviruses, measles viruses and retroviruses, cosmids or YACs, with plasmids being most preferred as vectors.

According to a preferred embodiment of the vector according to the invention, the DNA with a sequence as specified in any of groups a) to f) is under the control of a promoter capable of being regulated, which promoter is suitable for expressing the polypeptide encoded by these DNA sequences in the cell of a microorganism, preferably in a bacterial cell, a yeast cell or a fungal cell, especially preferably in a bacterial cell, most preferably in an E. Coli cell. Examples of such promoters are the trp promoter or the tac promoter.

Besides a promoter, the vector according to the invention should preferably comprise a ribosomal binding site and terminator. Here, it is especially preferred that the DNA according to the invention is incorporated into an expression cassette of the vector comprising the promoter, the ribosomal binding site and terminator. Besides the abovementioned structural elements, the vector may furthermore comprise selection genes known to the skilled worker.

A contribution to solving the problems mentioned at the outset is furthermore provided by the use of the above-described vector for transforming a cell and by the cell obtained by transformation of this vector. The cells which can be transformed with the vector according to the invention may be prokaryotes or eukaryotes. They may take the form of mammalian cells (such as, for example, cells from humans), of plant cells or of microorganisms such as yeasts, fungi or bacteria, with microorganisms being especially preferred and bacteria and yeasts being most preferred.

A contribution to solving the problems mentioned at the outset is also provided by a polypeptide which features the amino acid sequence with the SEQ ID No 04 or an amino acid sequence which is obtained when no more than 40 amino acids, preferably no more than 20 amino acids, even more preferably no more than 10 amino acids and most preferably no more than 5 amino acids in SEQ ID No 04 are deleted, inserted, substituted or else added to the C and/or N terminus of the amino acid sequence with the SEQ ID No 04. The polypeptide takes the form of an enzyme which is capable of catalyzing both the conversion of (S)- or (R)-methylmalonyl-coenzyme A into (S)- or (R)-methylmalonate semialdehyde and the conversion of malonyl-coenzyme A into malonate semialdehyde. Such a polypeptide can be obtained for example via the synthetic route, starting from the DNA sequence with the SEQ ID No 03, or by transformation of a suitable cell with a suitable vector comprising this nucleic acid sequence, expression, in the cell, of the protein encoded by this nucleic acid sequence, lysis of the cell, generating a cell extract, and subsequent purification of the enzyme by means of purification techniques known to the skilled worker, for example by means of HPLC or other chromatographic methods. Besides chromatographic purification of the polypeptide from cell extracts, one can also exploit the advantage that the polypeptide with the amino acid sequence SEQ ID No 04 is heat-resistant up to a temperature of at least 75° C. The cell extract can therefore be heated to a temperature of, for example, 75° C., which results in the coagulation, and thus precipitation, in the cell extract of those polypeptides which are not heat resistant. The polypeptide with the amino acid sequence SEQ ID No 04 is retained in the cell extract in nondenatured form.

EXAMPLES Example 1

The present invention is now illustrated in Example 1 with reference to a recombinant cell which is capable of producing 3-hydroxyisobutyric acid via 3-hydroxyiso-butyryl-coenzyme A as precursor and isobutyryl-coenzyme A as intermediate, starting from L-valine as carbon source. To this end, the enzymes EC 2.6.1.42 and EC 1.2.4.4 (in each case from Pseudomonas aeruginosa) and a cluster comprising the three enzymes EC 1.3.99.12, EC 4.2.1.17 and EC 3.1.2.4 (from Acinetobacter calcoaceticus) were overexpressed in E. coli BL21 (DE3).

Here, the enzyme EC 1.2.4.4 is encoded by a gene with the DNA sequence as shown in SEQ ID No 07 and 08 (α and β subunit), while the enzyme EC 2.6.1.42 is encoded by a gene with the DNA sequence as shown in SEQ ID No 09. The enzyme EC 1.3.99.12 is encoded by a gene with the DNA sequence with the SEQ ID No 10, the enzyme EC 4.2.1.17 by a gene with the DNA sequence as shown in SEQ ID No 11, and the enzyme EC 3.1.2.4 by a gene with the DNA sequence as shown in SEQ ID No 12.

1. Organisms, Plasmids and Oligonucleotides

-   -   The following bacterial strains, vectors, genomic DNA and         oligonucleotides were used for preparing this recombinant cell:

TABLE 1 Bacterial strains used Reference Strain (manufacturer) E. coli DH5 NEB E. coli BL21 (DE3) Invitrogen

TABLE 2 Vectors used Reference Vector (manufacturer) pCDFDuet-1 Novagen pET101/D-TOPO Invitrogen pCR2.1-TOPO Invitrogen

TABLE 3 Genomic DNA used Strain Pseudomonas aeruginosa PAO1 Acinetobacter calcoaceticus ADP1

TABLE 4 Oligonucleotides used Name Sequence Aca_VClus_fw 5′-ATGCAATTTAATGAAGAACAGCTATTAATTC- 3′ (SEQ ID No. 13) Aca_VClus_rev 5′-CAGTCTGAAATGACTAACCTAATTGGC-3′ (SEQ ID No. 14) Pae_26142_fw 5′-ACGGAATTCTGAAGGAGCTGGCAACTATG-3′ (SEQ ID No 15) Pae_26142_rev 5′-TTGTCGACTTACTTGACCAGGGTACGCC-3′ (SEQ ID No. 16) Pae_1244_fw 5′-ACAGATCTGGAGGCCTGTCATGAGTGATTAC- 3′ (SEQ ID No. 17) Pae_1244_rev 5′-ATGGGTACCCATTCAGACCTCCATC-3′ (SEQ ID No. 18)

2. Amplification of the PCR Fragments 1.2.4.4 (2313 kb) and 2.6.1.42 (958 bp)

-   -   First, the fragments of 1.2.4.4 and 2.6.1.42 were amplified by         means of PCR starting from the total DNA from Pseudomonas         aeruginosa, using the primers as shown in SEQ ID No 15 to SEQ ID         No 18, which are detailed in Table 4.

3. Digestion of the Vector pCDF-Duet-1 and of the PCR Fragment 2.6.1.42 (958 bp)

-   -   The vector pCDFDuet-1 (featuring a streptomycin-/spectinomycin         resistance) is cleaved by means of EcoRI/SalI, as is the PCR         fragment 2.6.1.42, and the restrictions thus obtained are         ligated overnight with T4 ligase. This gives rise to the vector         pCDFDuet::2.6.1.42.

4. Cloning of the PCR Fragments into the Vector pCR2.1-TOPO

-   -   The preparation of a cloning vector comprising the fragment         2.6.1.42 or the fragment 1.2.4.4, using the vector pCR2.1-TOPO,         was performed as specified in the manufacturer's         instructions. E. coli DH5α cells were transformed with the         resulting cloning vectors pCR2.1-TOPO::1.2.4.4 and         pCR2.1-TOPO::2.6.1.42. Since the pCR2.1-TOPO vectors feature a         kanamycin resistance and an ampicillin resistance, the         transformants were plated onto 2 AXI and KXI plates (20 and 40         μl). The plasmids of the resulting clones were isolated and         digested:     -   pCR2.1-TOPO::1.2.4.4 BgIII+KpnI fragment size 2313 bp     -   pCR2.1-TOPO::2.6.1.42 EcoRI+SalI fragment size 958 bp     -   Each of the fragments was eluted from the gel and purified with         the QIAquick kit from Qiagen (following instructions).

5. Preparation of the Vector pCDFDuet:2.6.1.42-1.2.4.4

-   -   The vector pCDFDuet::2.6.1.42 and the vector         pCR2.1-TOPO::1.2.4.4 are digested with BgIII/KpnI.

This is followed by the ligation of pCDFDuet::2.6.1.42 (BgIII/KpnI) with pCR2.1-TOPO::1.2.4.4, giving rise to the vector pCDFDuet::2.6.1.42-1.2.4.4. Again, E. coli DH5α cells were transformed by means of this cloning vector. The plasmids were isolated. The plasmid pCDFDuet::2.6.1.42-1.2.4.4 features the DNA sequence as shown in SEQ ID No 19.

6. Cloning the Valine Cluster from Acinetobacter calcoaceticus (V-Clus_(Aca))

-   -   Strain ATCC 33304 Acinetobacter calcoaceticus was cultured for         the isolation of total DNA (HH agar or medium). Total DNA was         isolated by means of the DNEasy kit from Qiagen (L1 and L2) and         by a method comprising the method steps i) centrifugation of 1         ml of culture, ii) addition of 200 μl of H₂O to the pellet, iii)         heating for 10 min at 95° C., iv) centrifugation (10 min, 13 000         rpm), and v) removing the supernatant for a PCR.     -   To amplify the valine cluster from A. calcoaceticus, a PCR was         carried out using the primers as shown in SEQ ID No 13 and SEQ         ID No 14, which have been detailed in Table 4 (following the         manufacturer's instructions using the polymerases Pfu and Taq,         respectively).     -   The PCR products were purified and, following the instructions,         ligated to the plasmid pET101/D-TOPO and transferred into E.         coli DH5α. This gives rise to the plasmid         pET101/D-TOPO::V-Cluster_(ACA). Plasmid         pET101/D-TOPO::V-Cluster_(Aca) features the DNA sequence as         shown in SEQ ID No 20.

7. Preparation of a Recombinant Cell which is Capable of Forming 3-Hydroxyisobutyric Acid from L-Valine

-   -   E. coli BL21 (DE3) was transformed with the plasmids         pET101/D-TOPO::V-Cluster_(Aca)and pCDF-Duet::2.6.1.42-1.2.4.4         (plated onto LB spec./amp medium). The resulting cells were         capable of converting, in a nutrient medium comprising L-valine,         the L-valine into 3-hydroxyisobutyric acid. In contrast, the         wild type of the cells (E. coli BL21 (DE3)) was not capable of         forming detectable amounts of 3-hydroxyisobutyric acid in such a         nutrient medium.

Example 2

In this example, the DNA according to the invention is isolated and the gene is overexpressed in E. coli.

1. Culturing and Harvesting Sulfolobus tokodaii

-   -   Sulfolobus tokodaii was grown in a small culture volume         (40-200 ml) at 75° C. and a pH of 3.0, with shaking (150 rpm).         The growth was monitored photometrically via measuring the         optical density at 578 nm (OD_(578 nm)). A modified Sulfolobus         medium was used (modified as described by Brock et al., Archives         of Microbiology 84, pages 54-68, 1972; Suzuki et al.,         Extremophiles, 6, pages 39-44, 2002). The energy and         carbohydrate source used were yeast extract, casamino acids and         glucose. The medium consisted of the following components: basal         medium, glucose stock solution, iron stock solution and trace         element stock solution. At an OD_(578nm) of 0.3-0.5 (exponential         phase), the cells were harvested. The centrifugation was carried         out in a Sorvall centrifuge (SS34 rotor) for 15 min at 9000 rpm.         The cell pellet was employed directly for the DNA extraction.         -   Basal medium. KH₂PO₄ (0.28 g/l, (NH₄)₂SO₄ (1.3 g/l), MgSO₄×7             H₂O (0.25 g/l), CaCl₂×6 H₂O (0.07 g/l), yeast extract (1             g/l) and casamino acids (1 g/l). Before autoclaving, the pH             was brought to 3.0 using H₂SO₄.         -   Glucose stock solution (100×). Glucose (100 g/l).         -   The solution was filter-sterilized.         -   Iron stock solution (1000×). FeCl₃×6 H₂O (20 g/l). The             solution was filter-sterilized.         -   Trace element stock solution (1000×). MnCl₂×4 H₂O (1.8 g/l),             Na₂B₄O₇×10 H₂O (4.5 g/l), ZnSO₄×7 H₂O (220 mg/l), CuCl₂×2             H₂O (50 mg/l), Na₂MoO₄×2 H₂O (30 mg/l), VOSO₄×5 H₂O (30             mg/l), CoCl₂×6 H₂O (8.4 mg/l). The individual components             were dissolved in succession in distilled H₂O, the pH was             brought to 3.0 using HCl, and the solution was             filter-sterilized.

2. Isolation of Genomic DNA from S. tokodaii

-   -   Genomic DNA was isolated by the method of Murray and Thompson         (Nucleic Acid Research, 8, pages 4321-4325, 1980). To this end,         10-50 mg (fresh weight) of freshly harvested cells are weighed         into a 1.5 ml Eppendorf reaction vessel and resuspended in 570         ml of TE buffer (10 mM Tris/HCl (pH 8.0), 1 mM NaEDTA). 30 μl of         a 10% (w/v) SDS solution (sodium dodecyl sulfate solution) and 3         μl of Proteinase K (20 μg/μl) were added and the mixture was         incubated for 1 h at 52° C. Thereafter, 100 μl of 5 M NaCl         solution and 80 μl of pre-warmed 10% (w/v)         cetyltrimethylammonium bromide (CTAB) solution (10% (w/v) CTAB         in 0.7 M NaCl) were added. After incubation for 10 min at 65°         C., the complexes of CTAB, cell wall fragments and proteins were         extracted with 780 μl of chloroform/iso-amyl alcohol (24:1         (v/v)) and spun down for 15 min at 14 000 rpm. The aqueous top         phase was transferred into a fresh Eppendorf reaction vessel and         the extraction was repeated. After the aqueous phase was free         from pigments, it was covered with a layer of 400 μl of 100%         isopropanol. By carefully mixing the two phases, the chromosomal         DNA precipitated at the interface. Then, it was possible to fish         out the DNA with a drawn-out Pasteur pipette and washed in 200         μl of 70% ethanol. After recentrifugation (5 min, 14 000 rpm),         the supernatant was pipetted off and the DNA was dried for 2 h         at room temperature and finally dissolved in 100 μl of TE         buffer.

3. Amplification of the Malonyl-Coenzyme A Reductase Gene

-   -   The polymer chain reaction (PCR) (Mullis et al., Cold Spring         Harbor Symp. Quant. Biol., 51, pages 263-273, 1986) was employed         to amplify the malonyl-CoA reductase gene in a targeted fashion,         from the genomic Sulfolobus tokodaii DNA obtained in Example 2.         It was carried out in a thermocycler (Biometra, Göttingen).     -   A preparative PCR in which Pfu polymerase (Pfunds, Genaxxon) was         used, was employed. The Pfu polymerase contains a 3′-5′         exonuclease (“proofreading”) function.     -   The following primers were used:

5′-ATTATCCCATGGGGAGAACATTAAAAGC-3′ (“forward primer”; NcoI cleavage site is underlined; SEQ ID No 21) and 5′-CGGGATCCTTACTTTTCAATATATCC-3′ (“reverse primer”; BamHI cleavage site is underlined; SEQ ID No 22)

-   -   The reaction mixture detailed in Table 1 hereinbelow was         employed for the PCR reactions. The PCR was carried out as a hot         start PCR, i.e. the reaction mixture was incubated for 2 min at         95° C. before adding the Pfu polymerase. This was followed by 30         cycles of in each case 1 minute at 95° C., 1 minute at 45° C.         and 5 minutes at 72° C., followed by a last step of 30 seconds         at 45° C., 15 minutes at 72° C. and, finally, a pause at 6° C.

TABLE 1 Standard reaction mixtures (50 μl) for proofreading PCR with Pfu polymerase Composition μl/50 μl batch 10 × Pfu PCR 5 reaction buffer dNTP mix (2 mM per 5 nucleotide) Forward primer (2 μM) 12.5 Reverse primer (2 μM) 12.5 Chromosomal DNA 1 (10-50 ng) Pfu polymerase 2 (2.5 U/μl) dd-H₂O 12

-   -   A gene fragment with a length of 1.1 kb was obtained.

4. Cloning the Malonyl-Coenzyme a Reductase Gene

-   -   To clone the malonyl-coenzyme A reductase gene from Sulfolobus         tokodaii, the gene amplified in Example 3 was cloned         unspecifically with the vector pCR T7/CT-Topo (Invitrogen,         Karlsruhe), using the “pCR T7 Topo TA Expression Kit”         (Invitrogen, Karlsruhe). This was done following the         manufacturer's instructions.     -   To isolate the plasmid DNA, the plasmid DNA was prepared using         the “QIAprep Spin Plasmid Miniprep Kit” from Qiagen (Hilden)         following the manufacturer's instructions, starting from 5 ml         overnight cultures of transformed E. coli TOP10F′ cells.

5. Generation of an Expression Vector

-   -   To generate an expression vector comprising the malonyl-coenzyme         A reductase gene, the isolated cloning vector obtained in         Example 4 is subjected to restriction digestion with the         restriction enzymes NcoI and BamHI. To this end, 25-27 μl of         plasmid DNA (expression vector pTrc99A and pCR T7/CT-Topo         vector, respectively, with the incorporated malonyl-coenzyme A         reductase gene) are mixed thoroughly with 5 pa of a reaction         buffer (10×) and 2-3 μl of restriction enzyme (10 U/μl;         Fermentas, St. Leon-Rot). The reaction mixture was made up to 50         μl with distilled H₂O and incubated for 5 h at the temperature         specified by the manufacturer. An ethanol precipitation was         carried out before further use. To this end, the DNA was mixed         with 3 volumes of 100% ethanol and 0.1 volumes of 3 M sodium         acetate buffer (pH 5.3) and incubated for 2 h or overnight at         −80° C. After a centrifugation step (20 min, 14 000 rpm, 4° C.,         Eppendorf table-top centrifuge), the supernatant is removed         carefully, and the DNA was washed with 3 volumes of 70% (v/v)         ethanol. After 10 min incubation at room temperature, the         mixture was recentrifuged (10 min, 14 000 rpm, 4° C., Eppendorf         table-top centrifuge) and the supernatant was discarded. The DNA         was then dried for 1 hour at room temperature and subsequently         taken up in the desired volume of H₂O or TE buffer (10 mM         Tris/HCl (pH 8.0), 1 mM NaEDTA).     -   Then, alkaline phosphatase is used for removing the 5′-phosphate         groups of the linearized double-stranded vector. In this manner,         the cloning efficiency is increased since religation of the         vector is prevented. Calf intestinal alkaline phosphatase was         used for dephosphorylating the digested vector.     -   The dephosphorylation was carried out in the same buffer as the         restriction digestion. 50 μl of restriction mixture were mixed         with 1.5 μl of CIAP (Calf Intestine Alkaline Phosphatase (1U/μl;         Fermentas, St. Leon-Rot) and the mixture was incubated for 30         min at 37° C. Before further use of the cleaved and         dephosphorylated vector, an ethanol precipitation was carried         out as described above.     -   T4 DNA ligase was used the ligation of the insert DNA with the         expression vector, plasmid DNA and insert DNA being employed in         a molar ratio of from 1:3-1:6.     -   Stock solutions:         -   Ligation buffer (10×): 0.5 M Tris/HCl, pH 7.6 100 mM MgCl₂             0.5 mg/ml BSA filter-sterilized, storage at room temperature         -   5 mM ATP (adenosine triphosphate) Always make up freshly in             sterile distilled H₂O         -   50 mM DTE (dithioerythritol) Always make up freshly in             ligation buffer     -   The ligation mixtures had a volume of 50 μl. Plasmid DNA (2-10         μl), insert DNA (2-20 μl), 5 μl of ligation buffer with DTE (50         mM) and the corresponding amount of sterile distilled H₂O were         pipetted together, vortexed, spun down briefly and subsequently         incubated for 5 min at 45° C. The mixture was cooled on ice. 5         μl of 5 mM ATP and 1.5 μl of T4 DNA ligase (1 U/μl; Fermentas;         St. Leon-Rot) were added, and everything was mixed. Ligation was         performed overnight at 16° C.     -   The ligation mixture was employed directly for transforming         chemically competent cells.

6. Transformation of E. Coli Cells with the Expression Vector

-   -   A 5 ml overnight culture was grown starting from a single colony         of E. coli Rosetta 2 cells. On the next morning, 50 ml of LB         medium (Sambrook et al., “Molecular Cloning: A Laboratory         Manual”, Cold Spring Harbor Laboratory Press, Cold Spring         Harbor, N.Y., 1989) were inoculated with 0.5-1.0 ml of this         culture. After incubation for 1.5-2 h (37° C., shaking (180         rpm)), an OD_(578 nm) of 0.6 was reached. The cells were cooled         on ice for 10 min and subsequently spun down for 5 min at 5000         rpm and 4° C. (GSA rotor, Sorvall centrifuge). The supernatant         was discarded and the cell pellet was resuspended in 2.7 ml of         cold 0.1 M CaCl₂ solution. After addition of 2.3 ml of sterile         50% (v/v) glycerol, the cell suspension was divided into         portions (in each case 300 μl) in 1.5-ml Eppendorf reaction         vessels. The competent cells were immediately frozen in liquid         nitrogen and subsequently stored at −80° C.     -   To transform the cells, an aliquot of the chemically competent         cells (300 μl) was defrosted on ice and treated with 25 μl of a         ligation mixture. Everything was mixed carefully and incubated         for 30 min on ice. After a heat shock (42° C., 1 min) the         mixture was reincubated on ice for 5 min. Thereafter, 800 μl of         LB medium (Sambrook et al., 1989) were added, and the cells were         shaken for 1 h at 37° C. (Thermomixer, Eppendorf 5436). The         mixture was concentrated and finally streaked onto LB medium. To         this end, the mixture was spun down for 1 min at 10 000 rpm, 750         μm of the supernatant were discarded, and the cell pellet was         resuspended. 50 μl, 100 μl and 200 μl of this concentrated         mixture were streaked onto LB plates (Sambrook et al., 1989)         supplemented with 100 μg/ml ampicillin and incubated overnight         in the incubator at 37° C. The plates were washed with 1 ml LB         medium. This cell suspension was used for subsequently         inoculating 150 ml LB medium (supplemented with 100 μg/ml         ampicillin) in 500 ml Erlenmeyer flasks with baffles. The         cultures grew at 37° C. and 180 rpm. Overexpression was         performed by inducing the promoter in pTrc99A by adding 0.5 M         IPTG (isopropyl-(β-D-thiogalactopyranoside) at an OD_(578nm) of         0.6. The induced cultures were incubated for 3 h under the         abovementioned conditions and subsequently harvested at an         OD_(578nm)=2.7.

7. Detection of the Enzymatic Activity

-   -   The E. coli strain obtained in Example 6 was disrupted by means         of a cell mill. The disrupted cells were heated for 15 min at         85° C. During this heat precipitation, nonheat resistant enzymes         coagulate and are precipitated. Since the target protein is heat         resistant, it is retained in the supernatant. To measure the         malonyl-coenzyme A reductase activity, the supernatant was         diluted 1:50 in TM buffer (50 mM Tris/Cl, 1 mM MgCl₂, pH 8.1).         30 μl of the diluted or undiluted (for detecting the         methylmalonyl-coenzyme A reductase activity) supernatant were         pipetted to 500 μl of HIPS buffer (100 mM HEPES/NaOH, 5 mM         MgCl₂, 1 mM dithioerythritol, containing 0.5 mM NADPH).     -   In a first batch, the reaction was started by adding         malonyl-coenzyme A, the final concentration being 0.5 mM. The         drop in the NADPH absorption at 365 nm was determined. The         enzyme activity determined was 15.5 μmol/min/mg protein (15.5         U/mg).     -   In a second batch, the reaction was started by adding         methylmalonyl-coenzyme A (from Fluka, Article No.: 67767), the         final concentration being 2.0 mM. The drop in the NADPH         absorption at 365 nm was determined. The enzyme activity         determined was 0.24 μmol/min/mg protein (0.24 U/mg).     -   It can be seen from these results that the polypeptide which         codes for the DNA sequence with the SEQ ID No 03 catalyzes both         the conversion of malonyl-CoA and of methylmalonyl-coenzyme A.     -   1 mol of NADPH was oxidized per mole of malonyl-CoA or         methylmalonyl-Coa employed. From this it can be concluded that         the enzymatic reaction leads to the corresponding semialdehyde. 

1. A cell which has been genetically modified in comparison with its wild type in such a way that it is capable of forming more 3-hydroxyisobutyric acid from L-valine, as a carbon source, in comparison with its wild type, wherein more 3-hydroxyisobutyric acid is formed from 3-hydroxyisobutyryl-coenzyme A as a precursor, wherein activities of enzymes E₈, E₆₀, E₆₁, E₇₉, and E₈₀ are increased in the cell compared to its wild type, wherein: E₈ is a 3-hydroxyisobutyryl-coenzyme A hydrolase EC 3.1.2.4, E₆₀ is an enoyl-coenzyme A hydratase EC 4.2.1.17, E₆₁ is a 2-methylacyl-coenzyme A dehydrogenase EC 1.3.99.12 E₇₉ is a 2-oxoisovalerate dehydrogenase EC 1.2.4.4, and E₈₀ is an amino acid transferase EC 2.6.1.42, wherein the cell is an Escherichia coli cell.
 2. A method of preparing a genetically modified cell which is capable of forming 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid using 3-hydroxybutyryl-coenzyme A, as a precursors, the method comprising increasing, in the cell, the activity of the enzymes of claim 1, wherein the cell is an Escherichia coli cell.
 3. A genetically modified cell obtainable by the method of claim
 2. 4. A method of producing 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid, the method comprising bringing a cell of claim 1 into contact with a nutrient medium comprising L-valine, as a carbon source, under conditions under which 3-hydroxyisobutyric acid or polyhydroxyalkanoates based on 3-hydroxyisobutyric acid are formed from the carbon source, and, optionally, purifying the 3-hydroxyisobutyric acid from the nutrient medium.
 5. A method of preparing methacrylic acid or methacrylic ester, the method comprising: IA) preparing 3-hydroxyisobutyric acid by the method of claim 4 and, optionally, neutralizing the 3-hydroxyisobutyric acid, and IB) dehydrating the 3-hydroxyisobutyric acid with formation of methacrylic acid and, optionally, esterifying the methacrylic acid.
 6. A method of preparing methacrylic acid or methacrylic esters, the method comprising: IIA) preparing a polyhydroxyalkanoate based on 3-hydroxybutyric acid by the method of claim 4, IIB) cleaving the polyhydroxyalkanoates with formation of 3-hydroxyisobutyric acid and, optionally, neutralizing the 3-hydroxyisobutyric acid, and IIC) dehydrating the 3-hydroxyisobutyric acid with formation of methacrylic acid and, optionally, esterifying the methacrylic acid.
 7. A method of preparing polymethacrylic acid or polymethacrylic esters, the method comprising: IIIA) preparing methacrylic acid by the method as claimed in claim 5, and IIIB) free-radical polymerizing the methacrylic acid.
 8. A method of preparing polymethacrylic acid or polymethacrylic esters, the method comprising: IIIA) preparing methacrylic acid by the method as claimed in claim 6, and IIIB) free-radical polymerizing the methacrylic acid. 